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.

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.

Production and Functional Characterization of a Recombinant Predicted Pore-Forming Protein (TVSAPLIP12) of Trichomonas vaginalis in Nicotiana benthamiana Plants.

Pore-forming proteins (PFPs) are a group of functionally versatile molecules distributed in all domains of life, and several microbial pathogens notably use members of this class of proteins as cytotoxic effectors. Among pathogenic protists, Entamoeba histolytica, and Naegleria fowleri display a range of pore-forming toxins belonging to the Saposin-Like Proteins (Saplip) family: Amoebapores and Naegleriapores. Following the genome sequencing of Trichomonas vaginalis, we identified a gene family of 12 predicted saposin-like proteins (TvSaplips): this work focuses on investigating the potential role of TvSaplips as cytopathogenetic effectors. We provide evidence that TvSaplip12 gene expression is potently upregulated upon T. vaginalis contact with target cells. We cloned and expressed recombinant TvSaplip12 in planta and we demonstrate haemolytic, cytotoxic, and bactericidal activities of rTvSaplip12 in vitro. Also, evidence for TvSaplip subcellular discrete distribution in cytoplasmic granules is presented. Altogether, our results highlight the importance of TvSaplip in T. vaginalis pathogenesis, depicting its involvement in the cytolytic and bactericidal activities during the infection process, leading to predation on host cells and resident vaginal microbiota for essential nutrients acquisition. This hence suggests a potential key role for TvSaplip12 in T. vaginalis pathogenesis as a candidate Trichopore.

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.

An Accessory Protease Inhibitor to Increase the Yield and Quality of a Tumour-Targeting mAb in Nicotiana benthamiana Leaves.

The overall quality of recombinant IgG antibodies in plants is dramatically compromised by host endogenous proteases. Different approaches have been developed to reduce the impact of endogenous proteolysis on IgGs, notably involving site-directed mutagenesis to eliminate protease-susceptible sites or the in situ mitigation of host protease activities to minimize antibody processing in the cell secretory pathway. We here characterized the degradation profile of H10, a human tumour-targeting monoclonal IgG, in leaves of Nicotiana benthamiana also expressing the human serine protease inhibitor α1-antichymotrypsin or the cysteine protease inhibitor tomato cystatin SlCYS8. Leaf extracts revealed consistent fragmentation patterns for the recombinant antibody regardless of leaf age and a strong protective effect of SlCYS8 in specific regions of the heavy chain domains. As shown using an antigen-binding ELISA and LC-MS/MS analysis of antibody fragments, SlCYS8 had positive effects on both the amount of fully-assembled antibody purified from leaf tissue and the stability of biologically active antibody fragments containing the heavy chain Fc domain. Our data confirm the potential of Cys protease inhibitors as convenient antibody-stabilizing expression partners to increase the quality of therapeutic antibodies in plant protein biofactories.

A biodistribution study of two differently shaped plant virus nanoparticles reveals new peculiar traits.

Self-assembling plant virus nanoparticles (pVNPs) have started to be explored as nanometre-sized objects for biomedical applications, such as vaccine or drug delivery and imaging. Plant VNPs may be ideal tools in terms of biocompatibility and biodegradability endowed with a wide diversity of symmetries and dimensions, easy chemical/biological engineering, and rapid production in plants. Recently, we defined that icosahedral Tomato bushy stunt virus (TBSV) and filamentous Potato virus X (PVX) are neither toxic nor teratogenic. We report here the results of an interdisciplinary study aimed to define for the first time the biodistribution of unlabelled, unpegylated, underivatized TBSV and PVX by proved detecting antibodies. These data add new insights on the in vivo behaviour of these nano-objects and demonstrate that the pVNPs under scrutiny are each intrinsically endowed with peculiar properties foreshadowing different applications in molecular medicine.

Production of a tumour-targeting antibody with a human-compatible glycosylation profile in N. benthamiana hairy root cultures.

Hairy root (HR) cultures derived from Agrobacterium rhizogenes transformation of plant tissues are an advantageous biotechnological manufacturing platform due to the accumulation of recombinant proteins in an otherwise largely protein free culture medium. In this context, HRs descending from transgenic Nicotiana tabacum plants were successfully used for the production of several functional mAbs with plant-type glycans. Here, we expressed the tumor-targeting monoclonal antibody mAb H10 in HRs obtained either by infecting a transgenic N. tabacum line expressing H10 with A. rhizogenes or a glyco-engineered N. benthamiana line (ΔXTFT) with recombinant A. rhizogenes carrying mAb H10 heavy and light chain cDNAs. Selected HR clones derived from both plants accumulated mAb H10 in the culture medium with similar yields (2-3 mg/L). N-glycosylation profiles of antibodies purified from HR supernatant revealed the presence of plant-typical complex structures for N. tabacum-derived mAb H10 and of GnGn structures lacking xylose and fucose for the ΔXTFT-derived counterpart. Both antibody glyco-formats exhibited comparable antigen binding activities. Collectively, these data demonstrate that the co-infection of ΔXTFT Nicotiana benthamiana with recombinant A. rhizogenes is an efficient procedure for the generation of stable HR cultures expressing the tumor-targeting mAb H10 with a human-compatible glycosylation profile, thus representing an important step towards the exploitation of root cultures for the production of 'next generation' human therapeutic antibodies.

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.

Proteome changes induced by c-myb silencing in human chronic myeloid leukemia cells suggest molecular mechanisms and putative biomarkers of hematopoietic malignancies.

To shed light on the molecular mechanisms associated with aberrant accumulation of c-Myb in chronic myeloid leukemia, comparative proteomic analysis was performed on c-myb RNAi-specifically silenced K562 cells, sampled on a time-course basis. 2D-DIGE technology highlighted 37 differentially-represented proteins that were further characterized by nLC-ESI-LIT-MS/MS and validated by western blotting and qRT-PCR analysis. Most of the deregulated proteins were related to protein folding, energy/primary metabolism, transcription/translation regulation and oxidative stress response. Protein network analysis suggested that glycolysis, gluconeogenesis and protein ubiquitination biosynthesis pathways were highly represented, confirming also the pivotal role of c-Myc. A specific reduced representation was observed for glyceraldehyde-3-phosphate-dehydrogenase and α-enolase, suggesting a possible role of c-Myb in the activation of aerobic glycolysis. A reduced amount was also observed for stress responsive heat shock 70kDa protein and 78kDa glucose-regulated protein, previously identified as direct targets of c-Myb. Among over-represented proteins, worth mentioning is the chromatin modifier chromobox protein homolog 3 that contributes to silencing of E2F- and Myc-responsive genes in quiescent G0 cells. Data here presented, while providing novel insights onto the molecular mechanisms underlying c-Myb activity, indicate potential protein biomarkers for monitoring the progression of chronic myeloid leukemia. BIOLOGICAL SIGNIFICANCE: Myeloid leukemia is a malignant disease of the hematopoietic system in which cells of myeloid lineages accumulate to an undifferentiated state. In particular, it was shown that an aberrant accumulation of the c-Myb transcriptional factor is associated with the suppression of normal differentiation processes promoting the development of the hematopoietic malignancies. Many efforts have been recently made to identify novel genes directly targeted by c-Myb at a transcriptome level. In this work, we originally describe a differential proteomic approach to facilitate the comprehension of the regulation of the protein networks exerted by c-Myb. Our study reveals a complex network of proteins regulated by c-Myb. The functional heterogeneity of these proteins emphasizes the pleiotropic role of c-Myb as a regulator of genes that are crucial for energy production and stress response in leukemia. In fact, variations in glyceraldehyde-3-phosphate-dehydrogenase and α-enolase suggest a possible role of c-Myb in the activation of aerobic glycolysis. Moreover, significant differences were found for heat shock 70kDa protein and 78kDa glucose-regulated protein known as direct c-Myb targets. This work highlights potential protein biomarkers to look into disease progression and to develop translational medicine approaches in myeloid leukemia.

Structure-based design and experimental engineering of a plant virus nanoparticle for the presentation of immunogenic epitopes and as a drug carrier.

Biomaterials research for the discovery of new generation nanoparticles is one of the most active areas of nanotechnology. In the search of nature-made nanometer-sized objects, plant virus particles appear as symmetrically defined entities that can be formed by protein self-assembly. In particular, in the field of plant virology, there is plenty of literature available describing the exploitation of plant viral cages to produce safe vaccine vehicles and nanoparticles for drug delivery. In this context, we have investigated on the use of the artichoke mottled crinkle virus (AMCV) capsid both as a carrier of immunogenic epitopes and for the delivery of anticancer molecules. A dual approach that combines both in silico tools and experimental virology was applied for the rational design of immunologically active chimeric virus-like particles (VLPs) carrying immunogenic peptides. The atomic structures of wild type (wt) and chimeric VLPs were obtained by homology modeling. The effects of insertion of the HIV-1 2F5 neutralizing epitope on the structural stability of chimeric VLPs were predicted and assessed by detailed inspection of the nanoparticle intersubunit interactions at atomic level. Wt and chimeric VLPs, exposing on their surface the 2F5 epitope, were successfully produced in plants. In addition, we demonstrated that AMCV capsids could also function as drug delivery vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first systematic predictive and empirical research addressing the question of how this icosahedral virus can be used for the production of both VLPs and viral nanoparticles for biomedical applications.

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.

Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants.

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.

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.

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.

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.

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).