Eugenio Benvenuto

Chimeric Plant Virus Particles as Immunogens for Inducing Murine and Human Immune Responses against Human Immunodeficiency Virus Type 1

ABSTRACT The high-yield expression of a neutralizing epitope from human immunodeficiency virus type 1 (HIV-1) on the surface of a plant virus and its immunogenicity are presented. The highly conserved ELDKWA epitope from glycoprotein (gp) 41 was expressed as an N-terminal translational fusion with the potato virus X (PVX) coat protein. The resulting chimeric virus particles (CVPs), purified and used to immunize mice intraperitoneally or intranasally, were able to elicit high levels of HIV-1-specific immunoglobulin G (IgG) and IgA antibodies. Furthermore, the human immune response to CVPs was studied with severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID). hu-PBL-SCID mice immunized with CVP-pulsed autologous dendritic cells were able to mount a specific human primary antibody response against the gp41-derived epitope. Notably, sera from both normal and hu-PBL-SCID mice showed an anti-HIV-1-neutralizing activity. Thus, PVX-based CVPs carrying neutralizing epitopes can offer novel perspectives for the development of effective vaccines against HIV and, more generally, for the design of new vaccination strategies in humans.

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.

Transient expression systems for plant-derived biopharmaceuticals.

In the molecular farming area, transient expression approaches for pharmaceutical proteins production, mainly recombinant monoclonal antibodies and vaccines, were developed almost two decades ago and, to date, these systems basically depend on Agrobacterium-mediated delivery and virus expression machinery. We survey here the current state-of-the-art of this research field. Several vectors have been designed on the basis of DNA- and RNA-based plant virus genomes and viral vectors are used both as single- and multicomponent expression systems in different combinations depending on the protein of interest. The obvious advantages of these systems are ease of manipulation, speed, low cost and high yield of proteins. In addition, Agrobacterium-mediated expression also allows the production in plants of complex proteins assembled from subunits. Currently, the transient expression methods are preferential over any other transgenic system for the exploitation of large and unrestricted numbers of plants in a contained environment. By designing optimal constructs and related means of delivery into plant cells, the overall technology plan considers scenarios that envisage high yield of bioproducts and ease in monitoring the whole spectrum of upstream production, before entering good manufacturing practice facilities. In this way, plant-derived bioproducts show promise of high competitiveness towards classical eukaryotic cell factory systems.

Strawberry proteome characterization and its regulation during fruit ripening and in different genotypes

Strawberry is worldwide appreciated for its unique flavour and as a source of macronutrients and high levels of antioxidants which are closely related to fruit ripening. We report the investigation of the complex physiological processes of strawberry fruit ripening at proteomic level. Multiple approaches were used to investigate strawberry fruit proteome. In particular, a proteome reference map of strawberry fruit from Queen Elisa élite genotype was achieved by 2-D analyses of proteins extracted from berries at immature, turning and red stages to isolate a set of proteins commonly present in fruit during ripening. In addition, several hundreds of proteins were identified by a combination of multidimensional liquid chromatography/tandem mass spectrometry and one dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry. DIGE technology was also used to identify differentially accumulated proteins during ripening and to correlate fruit protein expression with quality traits of the reference variety Queen Elisa and its parental genotypes. A number of constitutive or differentially accumulated proteins were found. Generally, the pattern of protein expression as well as the putative function of identified proteins argues for a role in major fruit physiological developmental and ripening processes. The role of some of the identified proteins is discussed in relation to strawberry fruit ripening and to quality traits. Consequently, this study provides the first characterization of the strawberry fruit proteome and the time course of variation during maturation by using multiple approaches.

‘Phytoantibodies’: a general vector for the expression of immunoglobulin domains in transgenic plants

Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium. The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immuno-histochemical analysis. Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction. It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

Plant pharming of a full‐sized, tumour‐targeting antibody using different expression strategies

The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.

New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and Bougainvillea spectabilis Willd.

Abstract. New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10−10 M range, and by various cell lines, with IC50s in the 10−8–10−6 M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237–282) with an LD50 (concentration that is 50% lethal) ≤ 8 mg · kg−1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 >32 mg · kg−1. The N-terminal sequence of the two RIPs from Basella rubra had 80–93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.

The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin

Protein bodies (PB) are stable polymers naturally formed by certain seed storage proteins within the endoplasmic reticulum (ER). The human immunodeficiency virus negative factor (Nef) protein, a potential antigen for the development of an anti-viral vaccine, is highly unstable when introduced into the plant secretory pathway, probably because of folding defects in the ER environment. The aim of this study was to promote the formation of Nef-containing PB in tobacco (Nicotiana tabacum) leaves by fusing the Nef sequence to the N-terminal domains of the maize storage protein γ-zein or to the chimeric protein zeolin (which efficiently forms PB and is composed of the vacuolar storage protein phaseolin fused to the N-terminal domains of γ-zein). Protein blots and pulse–chase indicate that fusions between Nef and the same γ-zein domains present in zeolin are degraded by ER quality control. Consistently, a mutated zeolin, in which wild-type phaseolin was substituted with a defective version known to be degraded by ER quality control, is unstable in plant cells. Fusion of Nef to the entire zeolin sequence instead allows the formation of PB detectable by electron microscopy and subcellular fractionation, leading to zeolin–Nef accumulation higher than 1% of total soluble protein, consistently reproduced in independent transgenic plants. It is concluded that zeolin, but not its γ-zein portion, has a positive dominant effect over ER quality control degradation. These results provide insights into the requirements for PB formation and avoidance of quality-control degradation, and indicate a strategy for enhancing foreign protein accumulation in plants.

Plant-produced potato virus X chimeric particles displaying an influenza virus-derived peptide activate specific CD8+ T cells in mice

Plant viruses can be genetically modified to produce chimeric virus particles (CVPs) carrying heterologous peptides. The efficacy of plant-produced CVPs in inducing antibody responses specific to the displayed peptide has been extensively demonstrated. To determine if plants can be used to produce CVPs able to activate peptide-specific major histocompatibility complex (MHC) class I-restricted CD8+ T cells, potato virus X (PVX) has been engineered to display the H-2D(b)-restricted epitope ASNENMETM of influenza A virus nucleoprotein (NP). Engineering criteria were devised to comply not only with plant virus genetic stability and infectivity but also with antigen processing rules. The immunological properties of different doses of endotoxin-free preparations of CVPs or unmodified PVX have been evaluated by s.c. immunizing C57BL/6J mice and testing at different time intervals splenocyte responses by interferon gamma (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay. These experiments demonstrated that CVPs activate ASNENMTEM-specific CD8+ T cells. Remarkably, the best response was achieved without adjuvant co-delivery. These results represent the proof of concept that well-designed plant virus carriers of epitopes produced in plant can reasonably be used into peptide vaccine formulations aimed to activate cell-mediated immune responses.

Genetic transformation of potato (Solanum tuberosum): An efficient method to obtain transgenic plants

A quick procedure for efficient transformation of potato (cv. Desiree) has been devised. Leaf disc have been inoculated with an Agrobacterium tumefaciens harbouring a Ti plasmid derived binary vector. Transformed shoots carrying the neomycin phosphotransferase gene were regenerated using a feeder layer technique after 4 weeks on selective medium containing kanamycin. Transgenic plants obtained in large numbers appeared phenotypically normal and expressed the NPT II gene. The present work points out that culture conditions are fundamental to obtain the highest transformation efficiency.