Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper.

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.

Tobacco seeds as efficient production platform for a biologically active anti-HBsAg monoclonal antibody.

The use of plants as heterologous hosts is one of the most promising technologies for manufacturing valuable recombinant proteins. Plant seeds, in particular, constitute ideal production platforms for long-term applications requiring a steady supply of starting material, as they combine the general advantages of plants as bioreactors with the possibility of biomass storage for long periods in a relatively small volume, thus allowing manufacturers to decouple upstream and downstream processing. In the present work we have used transgenic tobacco seeds to produce large amounts of a functionally active mouse monoclonal antibody against the Hepatitis B Virus surface antigen, fused to a KDEL endoplasmic reticulum retrieval motif, under control of regulatory sequences from common bean (Phaseolus vulgaris) seed storage proteins. The antibody accumulated to levels of 6.5 mg/g of seed in the T3 generation, and was purified by Protein A affinity chromatography combined with SEC-HPLC. N-glycan analysis indicated that, despite the KDEL signal, the seed-derived plantibody bore both high-mannose and complex-type sugars that indicate partial passage through the Golgi compartment, although its performance in the immunoaffinity purification of HBsAg was unaffected. An analysis discussing the industrial feasibility of replacing the currently used tobacco leaf-derived plantibody with this seed-derived variant is also presented.

A Plant-Derived Multi-HIV Antigen Induces Broad Immune Responses in Orally Immunized Mice.

Multi-HIV, a multiepitopic protein derived from both gp120 and gp41 envelope proteins of the human immunodeficiency virus (HIV), has been proposed as a vaccine prototype capable of inducing broad immune responses, as it carries various B and T cell epitopes from several HIV strains. In this study, the immunogenic properties of a Multi-HIV expressed in tobacco chloroplasts are evaluated in test mice. BALB/c mice orally immunized with tobacco-derived Multi-HIV have elicited antibody responses, including both the V3 loop of gp120 and the ELDKWA epitope of gp41. Based on splenocyte proliferation assays, stimulation with epitopes of the C4, V3 domain of gp120, and the ELDKWA domain of gp41 elicits positive cellular responses. Furthermore, specific interferon gamma production is observed in both CD4+ and CD8+ T cells stimulated with HIV peptides. These results demonstrate that plant-derived Multi-HIV induces T helper-specific responses. Altogether, these findings illustrate the immunogenic potential of plant-derived Multi-HIV in an oral immunization scheme. The potential of this low-cost immunization approach and its implications on HIV/AIDS vaccine development are discussed.

Comparative in vitro and experimental in vivo studies of the anti-epidermal growth factor receptor antibody nimotuzumab and its aglycosylated form produced in transgenic tobacco plants.

A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.

Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives.

Chlamydomonas reinhardtii has many advantages compared with traditional systems for the molecular farming of recombinant proteins. These include low production costs, rapid scalability at pilot level, absence of human pathogens and the ability to fold and assemble complex proteins accurately. Currently, the successful expression of several proteins with pharmaceutical relevance has been reported from the nuclear and the chloroplastic genome of this alga, demonstrating its usefulness for biotechnological applications. However, several factors affect the level of recombinant protein expression in Chlamydomonas such as enhancer elements, codon dependency, sensitivity to proteases and transformation-associated genotypic modification. The present review outlines a number of strategies to increase protein yields and summarizes recent achievements in algal protein production including biopharmaceuticals such as vaccines, antibodies, hormones and enzymes with implications on health-related approaches. The current status of bioreactor developments for algal culture and the challenges of scale-up and optimization processes are also discussed.

Differential N-glycosylation of a monoclonal antibody expressed in tobacco leaves with and without endoplasmic reticulum retention signal apparently induces similar in vivo stability in mice.

Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.

Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants.

Plants synthesize N-glycans containing the antigenic sugars alpha(1,3)-fucose and beta(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluated several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of alpha(1,3)-fucose and beta(1,2)-xylose. Ammonium hydroxide/carbonate-based chemical deglycosylation and PNGase A enzymatic release were investigated giving similar 2-aminobenzamide-labeled N-glycan HPLC profiles. The chemical release does not generate peptides which is convenient for MS analysis of unlabeled pool but its main drawback is that it induces degradation of alpha1,3-fucosylated N-glycan reducing terminal sugar. Three analytical methods for N-glycan characterization were evaluated: (i) MALDI-MS of glycopeptides from tryptic digestion; (ii) negative-ion ESI-MS/MS of released N-glycans; (iii) normal-phase HPLC of fluorescently labeled glycans in combination with exoglycosidase sequencing. The MS methods identified the major glycans, but the HPLC method was best for identification and relative quantitation of N-glycans. Negative-mode ESI-MS/MS permitted also the correct identification of the linkage position of the fucose residue linked to the inner core N-acteylglucosamine (GlcNAc) in complex N-glycans.

Production of plantibodies in Nicotiana plants.

Because of the wide use and high demand in medicine, monoclonal antibodies are among the main recombinant pharmaceuticals at present, although present limitations of the productive platforms for monoclonal antibodies are driving the improvement of the large-scale technologies and the development of alternative expression systems. This has drawn the attention on plants as expression system for monoclonal antibodies and related derivatives, owning the capacity of plants to properly express and process eukaryotic proteins with biological activity resembling that of the natural proteins. In this chapter, the procedures from the isolation of the monoclonal antibody genes to the biochemical and biological characterization of the plant-expressed monoclonal antibody are described.

Contenido de polifenoles y actividad antioxidante de varias semillas y nueces / Polyphenol content and antioxidant activity of several seeds and nuts

Los alimentos de origen vegetal en especial las frutas y los vegetales presentes en la dieta de acuerdo a estudios epidemiológicos realizados, pueden ejercer un efecto protector contra algunas enfermedades tales como el cáncer y trastornos cardiovasculares. Esta propiedad se debe a la presencia de compuestos bioactivos con capacidad antioxidante como la vitamina C, E, b-caroteno, y una mezcla compleja de compuestos fenólicos. El objetivo de este trabajo fue estudiar en una serie de productos de origen vegetal, la relación entre el contenido de polifenoles totales y la actividad antioxidante. Los polifenoles fueron determinados luego de su extracción en solución metanólica por el método de Folin-Ciocalteau. La actividad antioxidante fue evaluada usando los métodos del b-caroteno/linoleato, el poder reductor, y la actividad antirradical. Los productos estudiados fueron las semillas y/o pericarpios de: Theobroma cacao (cacao), Campsiandra comosa Benth (chiga), Sorghum bicolor, L. Moench (sorgo), Melicoccus bijugatus (mamón). El pericarpio del mamón presentó el más bajo contenido de polifenoles (1,40 EAGg/100g) y el cacao el más alto (6,66 EAGg/100g). El poder reductor del cacao resultó ser el más alto y equivalente al poder reductor de 5,80g de ácido ascórbico /100g, seguido por la chiga. Asimismo, las semillas de chiga y de cacao presentaron una actividad antioxidante, comparable a la del butil hidroxianisol antioxidante sintético. El mayor poder antirradical lo presentó la semilla de chiga con un EC50 de 2,67 g/gDPPH. El contenido de polifenoles totales se correlaciona bien con la actividad antioxidante; asimismo, estas semillas o granos podrían tener los efectos beneficiosos para la salud atribuidos a otras frutas y vegetales.

Foods from plant origin not only provide human diet with certain antioxidant vitamins (C, E and β-carotene), but also a complex mixture of polyphenols, with antioxidant activity. Numerous studies have been focused on the protective and preventing effect of this antioxidant activity on certain degenerative illnesses such as cardiovascular, cancer, and neurological diseases, cataracts and oxidative stress dysfunctions. The objective of this work was to evaluate total polyphenol content and antioxidant activity of several seeds, nuts, or grains such as Theobroma cacao, Campsiandra comosaBenth (chiga), Sorghum bicolor, L. Moench, Melicoccus bijugatus (genip). Total polyphenol content was assessed by the Folin-Ciocalteau method and the antioxidant activity by the β.carotene/linoleate, reducing power, and the anti-radical activity methods. Results showed genip pericarp with the lowest polyphenol content (1.40 gGAE/100g), and cacao beans with the highest (6.66 gGAE/100g). Reducing power of cacao beans was also the highest and similar to the reducing power of 5,80g ascorbic acid /100g, followed by Campsiandra comosa. Moreover, Campsiandra comosa and cacao seeds presented an antioxidant activity comparable to that of the butylhydroxianisol, a synthetic antioxidant. The highest anti-radical activity was shown by Campsiandra comosa with an EC50 of 2.67 g/gDPPH. Total polyphenol content shows a good correlation with the antioxidant activity. Moreover, these seeds might have the same health beneficial effects attributed to other fruits and vegetables.

Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco.

Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.

Comparison of different ligand densities in immunoaffinity chromatography of the plantibody HB-01 coupled to Sepharose CL-4B to purify the rHBsAg.

This paper evaluates the immunopurification behavior of a plantibody HBsAg specific plantibody coupled to Sepharose CL-4B at different ligand densities. Results show no significant differences in the adsorption and elution capacities, and rHBsAg recovery of immunosorbents at 3.43, 4.45, and 5.31 mg/mL of ligand densities compared to its mouse-derived mAb counterpart consistently used in the rHBsAg purification process. Therefore, plantibody ligand densities higher than 3.43 mg/mL do not improve the immunopurification behavior of this immunosorbent, but increase the antibody consumption and the Hepatitis B vaccine cost. Immunosorbent of 2.23 mg/mL of ligand density demonstrated a poor performance. The IgG leached detectable level never exceeded the approved limit (3 ng IgG/microg rHBsAg). Values close to this limit were only observed at the ligand density of 5.31 and 2.27 mg/mL. In the case of the ligand density of 2.23 mg/mL the IgG leached value was high (2.90 ng IgG/microg rHBsAg) due to a low level of eluted antigen. In conclusion, it supports feasibility of using this plantibody at 3.43 mg/mL of ligand density for large-scale immunopurification of rHBsAg for human use, avoiding the biosafety and ethical concerns of the massive use of animals for this purpose.

A rapid and sensitive ELISA to quantify an HBsAg specific monoclonal antibody and a plant-derived antibody during their downstream purification process.

An enzyme-linked immunosorbent assay to quantify the mAb CB.Hep-1 during downstream purification process was standardized and validated. This assay is characterized by a short time of incubation at high temperature, allowing the detection of this antibody with high specificity and sensitivity. Detection of antigen-antibody reaction was achieved using a horseradish peroxidase conjugated anti-mouse IgG whose enzyme activity was revealed with o-phenylenediamine substrate. The immunoassay is linear in a range between 3.12 and 50 ng/mL, with a recovery of 98.55-107.62%. According to results, it is possible to estimate the mAb CB.Hep-1 concentration with high precision and reproducibility. The intra- and interassay coefficient of variation ranged from 0.25 to 8.64% and 1.84 to 9.43%, respectively. Significant differences were not observed in the plant-derived antibody quantification by HRP-ELISA and PhoA-ELISA (n=18), demonstrating that plant endogenous peroxidases do not produce interferences in the quantification of this molecule. Therefore, both antibodies can be tested with the same immunoassay with high precision, specificity and accuracy during their respective purification processes without interference of the buffers and sample characteristics.