Luca Santi

Viral vectors for production of recombinant proteins in plants

Global demand for recombinant proteins has steadily accelerated for the last 20 years. These recombinant proteins have a wide range of important applications, including vaccines and therapeutics for human and animal health, industrial enzymes, new materials and components of novel nano‐particles for various applications. The majority of recombinant proteins are produced by traditional biological “factories,” that is, predominantly mammalian and microbial cell cultures along with yeast and insect cells. However, these traditional technologies cannot satisfy the increasing market demand due to prohibitive capital investment requirements. During the last two decades, plants have been under intensive investigation to provide an alternative system for cost‐effective, highly scalable, and safe production of recombinant proteins. Although the genetic engineering of plant viral vectors for heterologous gene expression can be dated back to the early 1980s, recent understanding of plant virology and technical progress in molecular biology have allowed for significant improvements and fine tuning of these vectors. These breakthroughs enable the flourishing of a variety of new viral‐based expression systems and their wide application by academic and industry groups. In this review, we describe the principal plant viral‐based production strategies and the latest plant viral expression systems, with a particular focus on the variety of proteins produced and their applications. We will summarize the recent progress in the downstream processing of plant materials for efficient extraction and purification of recombinant proteins. J. Cell. Physiol. 216: 366–377, 2008.

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

Production and characterization of an orally immunogenic Plasmodium antigen in plants using a virus-based expression system.

Increasing numbers of plant-made vaccines and pharmaceuticals are entering the late stage of product development and commercialization. Despite the theoretical benefits of such production, expression of parasite antigens in plants, particularly those from Plasmodium, the causative parasites for malaria, have achieved only limited success. We have previously shown that stable transformation of tobacco plants with a plant-codon optimized form of the Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5) gene resulted in PyMSP4/5 expression of up to approximately 0.25% of total soluble protein. In this report, we describe the rapid expression of PyMSP4/5 in Nicotiana benthamiana leaves using the deconstructed tobacco mosaic virus-based magnICON expression system. PyMSP4/5 yields of up to 10% TSP or 1-2 mg/g of fresh weight were consistently achieved. Characterization of the recombinant plant-made PyMSP4/5 indicates that it is structurally similar to PyMSP4/5 expressed by Escherichia coli. It is notable that the plant-made PyMSP4/5 protein retained its immunogenicity following long-term storage at ambient temperature within freeze-dried leaves. With assistance from a mucosal adjuvant the PyMSP4/5-containing leaves induced PyMSP4/5-specific antibodies when delivered orally to naïve mice or mice primed by a DNA vaccine. This study provides evidence that immunogenic Plasmodium antigens can be produced in large quantities in plants using the magnICON viral vector system.

Non‐food/feed seeds as biofactories for the high‐yield production of recombinant pharmaceuticals

We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.

Alcohol and wine in relation to cancer and other diseases.

Heavy alcohol consumption is associated with increased overall mortality, cancer, liver, and cardiovascular diseases; but low doses of alcohol (up to one drink per day) are not associated with the risk of any cancer site with the exception of breast cancer and possibly of oral and pharyngeal cancers. Moreover, recent evidence indicates that moderate alcohol and specifically wine intake provides cardioprotection and neuroprotection and may increase longevity. Various experimental data hypothesize a potential cancer chemopreventive role of some grape extracts, and complete sequencing of the grapevine genome has revealed genes responsible for the synthesis of health-promoting compounds (resveratrol and other polyphenols), thus advocating the development of future potential nutraceutical strategies. This focuses on the pros and cons of moderate alcohol and wine consumption and opens a debate on this topic.

Application of a multidimensional gas chromatography system with simultaneous mass spectrometric and flame ionization detection to the analysis of sandalwood oil

The production and trade of Indian sandalwood oil is strictly regulated, due to the impoverishment of the plantations; for such a reason, Australian sandalwood oil has been evaluated as a possible substitute of the Indian type. International directives report, for both the genuine essential oils, specific ranges for the sesquiterpene alcohols (santalols). In the present investigation, a multidimensional gas chromatographic system (MDGC), equipped with simultaneous flame ionization and mass spectrometric detection (FID/MS), has been successfully applied to the analysis of a series of sandalwood oils of different origin. A detailed description of the system utilized is reported. Three santalol isomers, (Z)-α-trans-bergamotol, (E,E)-farnesol, (Z)-nuciferol, epi-α-bisabolol and (Z)-lanceol have been quantified. LoD (MS) and LoQ (FID) values were determined for (E,E)-farnesol, used as representative of the oxygenated sesquiterpenic group, showing levels equal to 0.002% and 0.003%, respectively. A great advantage of the instrumental configuration herein discussed, is represented by the fact that identification and quantitation of target analytes are carried out in one step, without the need to perform two separate analyses.

Authentication of Bergamot Essential Oil by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometer (GC-C-IRMS)

Abstract The quality assessment of bergamot essential oils was established employing the gas chromatography-combustion- isotope ratio mass spectrometer (GC-C-IRMS) technique. An authenticity range was obtained investigating the carbon stable isotope ratio of genuine Italian bergamot essential oils (harvest period 2008–2009), in order to compare the GC-C-IRMS data of several industrial, commercial and foreign bergamot essential oil samples. Moreover, with the aim to test the efficiency and the sensibility of IRMS device, self-adulterated in laboratory bergamot oil samples were analyzed. The data were compared with those achieved by conventional enantioselective gas chromatography (Es-GC) and high resolution gas chromatography (GC-FID). Results of this work indicated that GC-C-IRMS was able not only to detect the presence of adulterants in the samples, but also to discriminate the bergamot oil samples according to their geographic provenance and the nature of the adulterants added.

Characterization of Oils from the Fruits, Leaves and Flowers of the Bitter Orange Tree

Abstract The bitter orange tree is used to obtain different products: essential oil cold pressed from the peel of the fruit, the neroli distilled from the flowers, the petitgrain distilled from the leaves and twigs, and different other products obtained by solvent extraction or by hydrodistillation. All these products are much appreciated for their odor properties. Their analytical characterization is important for the Identification of quality parameters, and considering their commercial value, it is important to determine their composition. This study is meant to determine differences between Italian and Egyptian cold-pressed, neroli and petitgrain oils, and gives useful information on the composition of the essential oil obtained from the whole crushed fruits. The composition of all the samples was studied by different analytical techniques to determine the volatile fraction, the enantiomeric distribution of some volatiles (by GC, GC/MS and esGC, respectively) and the oxygen heterocyclic fraction by RP-HPLC.

Plant-derived recombinant F1, V, and F1-V fusion antigens of Yersinia pestis activate human cells of the innate and adaptive immune system.

Plague is still endemic in different regions of the world. Current vaccines raise concern for their side effects and limited protection, highlighting the need for an efficacious and rapidly producible vaccine. F1 and V antigens of Yersinia pestis, and F1-V fusion protein produced in Nicotiana benthamiana administered to guinea pigs resulted in immunity and protection against an aerosol challenge of virulent Y. pestis. We examined the effects of plant-derived F1, V, and F1-V on human cells of the innate immunity. F1, V, and F1-V proteins engaged TLR2 signalling and activated IL-6 and CXCL-8 production by monocytes, without affecting the expression of TNF-α, IL-12, IL-10, IL-1β, and CXCL10. Native F1 antigen and recombinant plant-derived F1 (rF1) and rF1-V all induced similar specific T-cell responses, as shown by their recognition by T-cells from subjects who recovered from Y. pestis infection. Native F1 and rF1 were equally well recognized by serum antibodies of Y. pestis-primed donors, whereas serological reactivity to rF1-V hybrid was lower, and that to rV was virtually absent. In conclusion, plant-derived F1, V, and F1-V antigens are weakly reactogenic for human monocytes and elicit cell-mediated and humoral responses similar to those raised by Y. pestis infection.

Sicilian lemon oil: Composition of volatile and oxygen heterocyclic fractions and enantiomeric distribution of volatile components

A total of 92 samples of Sicilian lemon oils (Citrus limon (L.) Burm. f.) produced from September 2008 to June 2009, industrially cold-pressed by different extraction techniques (Brown Oil Extractor and Food Machinery Corporation), were analyzed by GC-flame ionization detector (FID) and GC/MS-LRI to investigate the volatile fraction; by RP-HPLC/PDA to determine the non-volatile components and by enantio-GC-FID to determine the enantiomeric ratios of 12 volatile components. This study provides a detailed investigation on the composition of Sicilian lemon essential oils industrially produced during a productive season, with the aim to recognize quality parameters for the characterization of this product. The results obtained are discussed to evaluate seasonal variation, influence of the extraction techniques, and are compared with those obtained for samples produced during different seasons.