Farming for Pharming: Novel Hydroponic Process in Contained Environment for Efficient Pharma-Grade Production of Saffron.

Soilless cultivation of saffron (Crocus sativus) in a controlled environment represents an interesting alternative to field cultivation, in order to obtain a standardized high-quality product and to optimize yields. In particular, pharma-grade saffron is fundamental for therapeutic applications of this spice, whose efficacy has been demonstrated in the treatment of macular diseases, such as Age-related Macular Degeneration (AMD). In this work, a hydroponic cultivation system was developed, specifically designed to meet the needs of C. sativus plant. Various cultivation recipes, different in spectrum and intensity of lighting, temperature, photoperiod and irrigation, have been adopted to study their effect on saffron production. The experimentation involved the cultivation of corms from two subsequent farm years, to identify and validate the optimal conditions, both in terms of quantitative yield and as accumulation of bioactive metabolites, with particular reference to crocins and picrocrocin, which define the 'pharma-grade' quality of saffron. Through HPLC analysis and chromatography it was possible to identify the cultivation parameters suitable for the production of saffron with neuroprotective properties, evaluated by comparison with an ISO standard and the REPRON® procedure. Furthermore, the biochemical characterization was completed through NMR and high-resolution mass spectrometry analyses of saffron extracts. The whole experimental framework allowed to establish an optimized protocol to produce pharma-grade saffron, allowing up to 3.2 g/m2 harvest (i.e., more than three times higher than field production in optimal conditions), which meets the standards of composition for the therapy of AMD.

Optimised production of an anti-fungal antibody in Solanaceae hairy roots to develop new formulations against Candida albicans.

BACKGROUND: Infections caused by fungi are often refractory to conventional therapies and urgently require the development of novel options, such as immunotherapy. To produce therapeutic antibodies, a plant-based expression platform is an attractive biotechnological strategy compared to mammalian cell cultures. In addition to whole plants, hairy roots (HR) cultures can be used, representing an expression system easy to build up, with indefinite growth while handled under containment conditions. RESULTS: In this study the production in HR of a recombinant antibody, proved to be a good candidate for human immunotherapy against fungal infections, is reported. Expression and secretion of this antibody, in an engineered single chain (scFvFc) format, by HR from Nicotiana benthamiana and Solanum lycopersicum have been evaluated with the aim of directly using the deriving extract or culture medium against pathogenic fungi. Although both Solanaceae HR showed good expression levels (up to 68 mg/kg), an optimization of rhizosecretion was only obtained for N. benthamiana HR. A preliminary assessment to explain this result highlighted the fact that not only the presence of proteases, but also the chemical characteristics of the growth medium, can influence antibody yield, with implications on recombinant protein production in HR. Finally, the antifungal activity of scFvFc 2G8 antibody produced in N. benthamiana HR was evaluated in Candida albicans growth inhibition assays, evidencing encouraging results. CONCLUSIONS: Production of this anti-fungal antibody in HR of N. benthamiana and S. lycopersicum elucidated factors affecting pharming in this system and allowed to obtain promising ready-to-use immunotherapeutics against C. albicans.

N-glycan engineering of a plant-produced anti-CD20-hIL-2 immunocytokine significantly enhances its effector functions.

Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N-glycosylation plant signature (IC-ΔXF) and the corresponding scFv-Fc-ΔXF antibody not fused to the cytokine, were obtained in a glyco-engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20-35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC-ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N-glycosylation profile (IC) and to rituximab. The glyco-engineered recombinant molecules also exhibited a strongly improved ADCC and complement-dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv-Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N-glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential.

Comparative analysis of plant-produced, recombinant dimeric IgA against cell wall ß-glucan of pathogenic fungi.

Immunoglobulins A (IgA) are crucially involved in protection of human mucosal surfaces from microbial pathogens. In this work, we devised and expressed in plants recombinant chimeric antifungal antibodies (Abs) of isotype A (IgA1, IgA2, and scFvFcA1), derived from a murine mAb directed to the fungal cell wall polysaccharide ß-glucan which had proven able to confer protection against multiple pathogenic fungi. All recombinant IgA (rIgA) were expressed and correctly assembled in dimeric form in plants and evaluated for yield, antigen-binding efficiency and antifungal properties in vitro, in comparison with a chimeric IgG1 version. Production yields and binding efficiency to purified ß-glucans showed significant variations not only between Abs of different isotypes but also between the different IgA formats. Moreover, only the dimeric IgA1 was able to strongly bind cells of the fungal pathogen Candida albicans and to restrain its adhesion to human epithelial cells. Our data indicate that IgG to IgA switch and differences in molecular structure among different rIgA formats can impact expression in plant and biological activity of anti-ß-glucans Abs and provide new insights for the design of recombinant IgA as anti-infective immunotherapeutics, whose potential is still poorly investigated.

Production of an active anti-CD20-hIL-2 immunocytokine in Nicotiana benthamiana.

Anti-CD20 murine or chimeric antibodies (Abs) have been used to treat non-Hodgkin lymphomas (NHLs) and other diseases characterized by overactive or dysfunctional B cells. Anti-CD20 Abs demonstrated to be effective in inducing regression of B-cell lymphomas, although in many cases patients relapse following treatment. A promising approach to improve the outcome of mAb therapy is the use of anti-CD20 antibodies to deliver cytokines to the tumour microenvironment. In particular, IL-2-based immunocytokines have shown enhanced antitumour activity in several preclinical studies. Here, we report on the engineering of an anti-CD20-human interleukin-2 (hIL-2) immunocytokine (2B8-Fc-hIL2) based on the C2B8 mAb (Rituximab) and the resulting ectopic expression in Nicotiana benthamiana. The scFv-Fc-engineered immunocytokine is fully assembled in plants with minor degradation products as assessed by SDS-PAGE and gel filtration. Purification yields using protein-A affinity chromatography were in the range of 15-20 mg/kg of fresh leaf weight (FW). Glycopeptide analysis confirmed the presence of a highly homogeneous plant-type glycosylation. 2B8-Fc-hIL2 and the cognate 2B8-Fc antibody, devoid of hIL-2, were assayed by flow cytometry on Daudi cells revealing a CD20 binding activity comparable to that of Rituximab and were effective in eliciting antibody-dependent cell-mediated cytotoxicity of human PBMC versus Daudi cells, demonstrating their functional integrity. In 2B8-Fc-hIL2, IL-2 accessibility and biological activity were verified by flow cytometry and cell proliferation assay. To our knowledge, this is the first example of a recombinant immunocytokine based on the therapeutic Rituximab antibody scaffold, whose expression in plants may be a valuable tool for NHLs treatment.

Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis.

Effects of gamma radiation on engineered tomato biofortified for space agriculture by morphometry and fluorescence-based indices.

Introduction: Future long-term space missions will focus to the solar system exploration, with the Moon and Mars as leading goals. Plant cultivation will provide fresh food as a healthy supplement to astronauts' diet in confined and unhealthy outposts. Ionizing radiation (IR) are a main hazard in outer space for their capacity to generate oxidative stress and DNA damage. IR is a crucial issue not only for human survival, but also for plant development and related value-added fresh food harvest. To this end, efforts to figure out how biofortification of plants with antioxidant metabolites (such as anthocyanins) may contribute to improve their performances in space outposts are needed. Methods: MicroTom plants genetically engineered to express the Petunia hybrida PhAN4 gene, restoring the biosynthesis of anthocyanins in tomato, were used. Seeds and plants from wild type and engineered lines AN4-M and AN4-P2 were exposed to IR doses that they may experience during a long-term space mission, simulated through the administration of gamma radiation. Plant response was continuously evaluated along life cycle by a non-disturbing/non-destructive monitoring of biometric and multiparametric fluorescence-based indices at both phenotypic and phenological levels, and indirectly measuring changes occurring at the primary and secondary metabolism level. Results: Responses to gamma radiation were influenced by the phenological stage, dose and genotype. Wild type and engineered plants did not complete a seed-to-seed cycle under the exceptional condition of 30 Gy absorbed dose, but were able to cope with 0.5 and 5 Gy producing fruits and vital seeds. In particular, the AN4-M seeds and plants showed advantages over wild type: negligible variation of fluorimetric parameters related to primary metabolism, no alteration or improvement of yield traits at maturity while maintaining smaller habitus than wild type, biosynthesis of anthocyanins and maintained levels of these compounds compared to non-irradiated controls of the same age. Discussion: These findings may be useful in understanding phenotypic effects of IR on plant growth in space, and lead to the exploitation of new breeding efforts to optimize plant performances to develop appropriate ideotypes for future long-term space exploration extending the potential of plants to serve as high-value product source.

Design of a modular controlled unit for the study of bioprocesses: Towards solutions for Bioregenerative Life Support Systems in space.

Space exploration beyond the Low Earth Orbit requires the establishment of Bioregenerative Life Support Systems (BLSSs), which, through bioprocesses for primary resource recycling, ensure crew survival. However, the introduction of new organisms in confined space habitats must be carefully evaluated in advance to avoid unforeseen events that could compromise the mission. In this work, we have designed and built an experimental chamber, named Growing/Rearing Module (GRM), completely isolated and equipped with micro-environmental monitoring and control systems. This unit is specially intended for the study of single bioprocesses, which can be composed to design functional BLSSs. GRM can be implemented with specific devices for the biological system under study and the control of environmental parameters such as temperature, humidity, lighting and if required, pressure of gaseous components. GRM was validated in experiments of both microgreen cultivation, as a source of fresh food for astronauts, and rearing of the decomposer insect Hermetia illucens for bioconversion of organic waste. During the study of each bioprocess, the environmental and biological data were recorded, allowing to make preliminary assessments of the system efficiency. The GRM, as a completely confined environment, represents the first self-consistent unit that allows to fine-tune the optimal parameters for the operability of different bioprocesses. Furthermore, the upgradability according to the mission needs and the functional integrability of modules differently equipped are the keys to GRM versatility, representing a valuable tool for BLSSs' design.

2-D DIGE analysis of UV-C radiation-responsive proteins in globe artichoke leaves.

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ∼144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.

Recent insights into plant-virus interactions through proteomic analysis.

Plant viruses represent a major threat for a wide range of host species causing severe losses in agricultural practices. The full comprehension of mechanisms underlying events of virus-host plant interaction is crucial to devise novel plant resistance strategies. Until now, functional genomics studies in plant-virus interaction have been limited mainly on transcriptomic analysis. Only recently are proteomic approaches starting to provide important contributions to this area of research. Classical two-dimensional electrophoresis (2-DE) coupled to mass spectrometry (MS) is still the most widely used platform in plant proteome analysis, although in the last years the application of quantitative "second generation" proteomic techniques (such as differential in gel electrophoresis, DIGE, and gel-free protein separation methods) are emerging as more powerful analytical approaches. Apparently simple, plant-virus interactions reveal a really complex pathophysiological context, in which resistance, defense and susceptibility, and direct virus-induced reactions interplay to trigger expression responses of hundreds of genes. Given that, this review is specifically focused on comparative proteome-based studies on pathogenesis of several viral genera, including some of the most important and widespread plant viruses of the genus Tobamovirus, Sobemovirus, Cucumovirus and Potyvirus. In all, this overview reveals a widespread repression of proteins associated with the photosynthetic apparatus, while energy metabolism/protein synthesis and turnover are typically up-regulated, indicating a major redirection of cell metabolism. Other common features include the modulation of metabolisms concerning sugars, cell wall, and reactive oxigen species as well as pathogenesis-related (PR) proteins. The fine-tuning between plant development and antiviral defense mechanisms determines new patterns of regulation of common metabolic pathways. By offering a 360-degree view of protein modulation, all proteomic tools reveal the extraordinary intricacy of mechanisms with which a simple viral genome perturbs the plant cell molecular networks. This "omic" approach, while providing a global perspective and useful information to the understanding of the plant host-virus interactome, may possibly reveal protein targets/markers useful in the design of future diagnosis and/or plant protection strategies.

Heat-shock protein 70 from plant biofactories of recombinant antigens activate multiepitope-targeted immune responses.

Although a physiological role of heat-shock proteins (HSP) in antigen presentation and immune response activation has not been directly demonstrated, their use as vaccine components is under clinical trial. We have previously demonstrated that the structure of plant-derived HSP70 (pHSP70) can be superimposed to the mammalian homologue and similarly to the mammalian counterpart, pHSP70-polypeptide complexes can activate the immune system. It is here shown that pHSP70 purified from plant tissues transiently expressing the influenza virus nucleoprotein are able to induce both the activation of major histocompatibility complex class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery. These results indicate that pHSP70 derived from plants producing recombinant antigens may be used to formulate multiepitope vaccines.

Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation.

We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which ß1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of ß1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible 'fragile' sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.

Modifying Anthocyanins Biosynthesis in Tomato Hairy Roots: A Test Bed for Plant Resistance to Ionizing Radiation and Antioxidant Properties in Space.

Gene expression manipulation of specific metabolic pathways can be used to obtain bioaccumulation of valuable molecules and desired quality traits in plants. A single-gene approach to impact different traits would be greatly desirable in agrospace applications, where several aspects of plant physiology can be affected, influencing growth. In this work, MicroTom hairy root cultures expressing a MYB-like transcription factor that regulates the biosynthesis of anthocyanins in Petunia hybrida (PhAN4), were considered as a testbed for bio-fortified tomato whole plants aimed at agrospace applications. Ectopic expression of PhAN4 promoted biosynthesis of anthocyanins, allowing to profile 5 major derivatives of delphinidin and petunidin together with pelargonidin and malvidin-based anthocyanins, unusual in tomato. Consistent with PhAN4 features, transcriptomic profiling indicated upregulation of genes correlated to anthocyanin biosynthesis. Interestingly, a transcriptome reprogramming oriented to positive regulation of cell response to biotic, abiotic, and redox stimuli was evidenced. PhAN4 hairy root cultures showed the significant capability to counteract reactive oxygen species (ROS) accumulation and protein misfolding upon high-dose gamma irradiation, which is among the most potent pro-oxidant stress that can be encountered in space. These results may have significance in the engineering of whole tomato plants that can benefit space agriculture.

Two-dimensional differential in gel electrophoresis (2D-DIGE) analysis of grape berry proteome during postharvest withering.

The practice of postharvest withering is commonly used to correct quality traits and sugar concentration of high quality wines. To date, changes in the metabolome during the berry maturation process have been well documented; however, the biological events which occur at the protein level have yet to be fully investigated. To gain insight into the postharvest withering process, we studied the protein expression profiles of grape (Corvina variety) berry development focusing on withering utilizing a two-dimensional differential in gel electrophoresis (2D-DIGE) proteomics approach. Comparative analysis revealed changes in the abundance of numerous soluble proteins during the maturation and withering processes. On a total of 870 detected spots, 90 proteins were differentially expressed during berry ripening/withering and 72 were identified by MS/MS analysis. The majority of these proteins were related to stress and defense activity (30%), energy and primary metabolism (25%), cytoskeleton remodelling (7%), and secondary metabolism (5%). Moreover, this study demonstrates an active modulation of metabolic pathways throughout the slow dehydration process, including de novo protein synthesis in response to the stress condition and further evolution of physiological processes originated during ripening. These data represent an important insight into the withering process in terms of both Vitis germplasm characterization and knowledge which can assist quality improvement.

LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies.

Oil bodies (OBs) are plant cell organelles that consist of a lipid core surrounded by a phospholipid monolayer embedded with specialized proteins such as oleosins. Recombinant proteins expressed in plants can be targeted to OBs as fusions with oleosin. This expression strategy is attractive because OBs are easily enriched and purified from other cellular components, based on their unique physicochemical properties. For recombinant OBs to be a potential therapeutic agent in biomedical applications, it is necessary to comprehensively analyze and quantify both endogenous and heterologously expressed OB proteins. In this study, a mass spectrometry (MS)-based method was developed to accurately quantify an OB-targeted heterologously expressed fusion protein that has potential as a therapeutic agent. The effect of the chimeric oleosin expression upon the OB proteome in transgenic plants was also investigated, and the identification of new potential OB residents was pursued through a variety of liquid chromatography (LC)-MS/MS approaches. The results showed that the accumulation of the fusion protein on OBs was low. Moreover, no significant differences in the accumulation of OB proteins were revealed between transgenic and wild-type seeds. The identification of five new putative components of OB proteome was also reported.

Plant production of anti-ß-glucan antibodies for immunotherapy of fungal infections in humans.

There is an increasing interest in the development of therapeutic antibodies (Ab) to improve the control of fungal pathogens, but none of these reagents is available for clinical use. We previously described a murine monoclonal antibody (mAb 2G8) targeting ß-glucan, a cell wall polysaccharide common to most pathogenic fungi, which conferred significant protection against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans in animal models. Transfer of this wide-spectrum, antifungal mAb into the clinical setting would allow the control of most frequent fungal infections in many different categories of patients. To this aim, two chimeric mouse-human Ab derivatives from mAb 2G8, in the format of complete IgG or scFv-Fc, were generated, transiently expressed in Nicotiana benthamiana plants and purified from leaves with high yields (approximately 50 mg Ab/kg of plant tissues). Both recombinant Abs fully retained the ß-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially ß1,3-linked glucan molecules present at the fungal cell surface and directly inhibited the growth of C. albicans and its adhesion to human epithelial cells in vitro. In addition, both the IgG and the scFv-Fc promoted C. albicans killing by isolated, human polymorphonuclear neutrophils in ex vivo assays and conferred significant antifungal protection in animal models of systemic or vulvovaginal C. albicans infection. These recombinant Abs represent valuable molecules for developing novel, plant-derived immunotherapeutics against candidiasis and, possibly, other fungal diseases.

Identification of putative stage-specific grapevine berry biomarkers and omics data integration into networks.

The analysis of grapevine (Vitis vinifera) berries at the transcriptomic, proteomic, and metabolomic levels can provide great insight into the molecular events underlying berry development and postharvest drying (withering). However, the large and very different data sets produced by such investigations are difficult to integrate. Here, we report the identification of putative stage-specific biomarkers for berry development and withering and, to our knowledge, the first integrated systems-level study of these processes. Transcriptomic, proteomic, and metabolomic data were integrated using two different strategies, one hypothesis free and the other hypothesis driven. A multistep hypothesis-free approach was applied to data from four developmental stages and three withering intervals, with integration achieved using a hierarchical clustering strategy based on the multivariate bidirectional orthogonal projections to latent structures technique. This identified stage-specific functional networks of linked transcripts, proteins, and metabolites, providing important insights into the key molecular processes that determine the quality characteristics of wine. The hypothesis-driven approach was used to integrate data from three withering intervals, starting with subdata sets of transcripts, proteins, and metabolites. We identified transcripts and proteins that were modulated during withering as well as specific classes of metabolites that accumulated at the same time and used these to select subdata sets of variables. The multivariate bidirectional orthogonal projections to latent structures technique was then used to integrate the subdata sets, identifying variables representing selected molecular processes that take place specifically during berry withering. The impact of this holistic approach on our knowledge of grapevine berry development and withering is discussed.

Plant heat shock protein 70 as carrier for immunization against a plant-expressed reporter antigen.

Mammalian Heat Shock Proteins (HSP), have potent immune-stimulatory properties due to the natural capability to associate with polypeptides and bind receptors on antigen presenting cells. The present study was aimed to explore whether plant HSP, and in particular HSP70, share similar properties. We wanted in particular to evaluate if HSP70 extracted in association to naturally bound polypeptides from plant tissues expressing a recombinant "reporter" antigen, carry antigen-derived polypeptides and can be used to activate antigen-specific immune responses. This application of HSP70 has been very poorly investigated so far. The analysis started by structurally modeling the plant protein and defining the conditions that ensure maximal expression levels and optimal recovery from plant tissues. Afterwards, HSP70 was purified from Nicotiana benthamiana leaves transiently expressing a heterologous "reporter" protein. The purification was carried out taking care to avoid the release from HSP70 of the polypeptides chaperoned within plant cells. The evaluation of antibody titers in mice sera subsequent to the subcutaneous delivery of the purified HSP70 demonstrated that it is highly effective in priming humoral immune responses specific to the plant expressed "reporter" protein. Overall results indicated that plant-derived HSP70 shares structural and functional properties with the mammalian homologue. This study paves the way to further investigations targeted at determining the properties of HSP70 extracted from plants expressing foreign recombinant antigens as a readily available immunological carrier for the efficient delivery of polypeptides derived from these antigens.

Proteomic analysis of the plant-virus interaction in cucumber mosaic virus (CMV) resistant transgenic tomato.

Cucumber mosaic virus (CMV), a member of the Cucumovirus genus, is the causal agent of several plant diseases in a wide range of host species, causing important economic losses in agriculture. Because of the lack of natural resistance genes in most crops, different genetic engineering strategies have been adopted to obtain virus-resistant plants. In a previous study, we described the engineering of transgenic tomato plants expressing a single-chain variable fragment antibody (scFv G4) that are specifically protected from CMV infection. In this work, we characterized the leaf proteome expressed during compatible plant-virus interaction in wild type and transgenic tomato. Protein changes in both inoculated and apical leaves were revealed using two-dimensional gel electrophoresis (2-DE) coupled to differential in gel electrophoresis (DIGE) technology. A total of 2084 spots were detected, and 50 differentially expressed proteins were identified by nanoscale liquid chromatographic-electrospray ionization-ion trap-tandem mass spectrometry (nLC-ESI-IT-MS/MS). The majority of these proteins were related to photosynthesis (38%), primary metabolism (18%), and defense activity (14%) and demonstrated to be actively down regulated by CMV in infected leaves. Moreover, our analysis revealed that asymptomatic apical leaves of transgenic inoculated plants had no protein profile alteration as compared to control wild type uninfected plants demonstrating that virus infection is confined to the inoculated leaves and systemic spread is hindered by the CMV coat protein (CP)-specific scFv G4 molecules. Our work is the first comparative study on compatible plant-virus interactions between engineered immunoprotected and susceptible wild type tomato plants, contributing to the understanding of antibody-mediated disease resistance mechanisms.

Optimisation of the purification process of a tumour-targeting antibody produced in N. benthamiana using vacuum-agroinfiltration.

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 µg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15-20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 µg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).