Expression, intracellular targeting and purification of HIV Nef variants in tobacco cells

Background Plants may represent excellent alternatives to classical heterologous protein expression systems, especially for the production of biopharmaceuticals and vaccine components. Modern vaccines are becoming increasingly complex, with the incorporation of multiple antigens. Approaches towards developing an HIV vaccine appear to confirm this, with a combination of candidate antigens. Among these, HIV-Nef is considered a promising target for vaccine development because immune responses directed against this viral protein could help to control the initial steps of viral infection and to reduce viral loads and spreading. Two isoforms of Nef protein can be found in cells: a full-length N-terminal myristoylated form (p27, 27 kDa) and a truncated form (p25, 25 kDa). Here we report the expression and purification of HIV Nef from transgenic tobacco. Results We designed constructs to direct the expression of p25 and p27 Nef to either the cytosol or the secretory pathway. We tested these constructs by transient expression in tobacco protoplasts. Cytosolic Nef polypeptides are correctly synthesised and are stable. The same is not true for Nef polypeptides targeted to the secretory pathway by virtue of a signal peptide. We therefore generated transgenic plants expressing cytosolic, full length or truncated Nef. Expression levels were variable, but in some lines they averaged 0.7% of total soluble proteins. Hexahistidine-tagged Nef was easily purified from transgenic tissue in a one-step procedure. Conclusion We have shown that transient expression can help to rapidly determine the best cellular compartment for accumulation of a recombinant protein. We have successfully expressed HIV Nef polypeptides in the cytosol of transgenic tobacco plants. The proteins can easily be purified from transgenic tissue

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

<p>Abstract</p> <p>Background</p> <p>In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in <it>Nicotiana benthamiana</it>, followed by a two-step affinity purification protocol of plant-derived Nef.</p> <p>Results</p> <p>The effect of three gene silencing viral suppressor proteins (P25 of Potato Virus X, P19 of either Artichoke Mottled Crinckle virus and Tomato Bushy Stunt virus) on Nef transient expression yield was evaluated. The P19 protein of Artichoke Mottled Crinckle virus (AMCV-P19) gave the highest expression yield in vacuum co-agroinfiltration experiments reaching 1.3% of total soluble protein, a level almost three times higher than that previously reported in stable transgenic plants. The high yield observed in the co-agroinfiltrated plants was correlated to a remarkable decrease of Nef-specific small interfering RNAs (siRNAs) indicating an effective modulation of RNA silencing mechanisms by AMCV-P19. Interestingly, we also showed that expression levels in top leaves of vacuum co-agroinfiltrated plants were noticeably reduced compared to bottom leaves. Moreover, purification of Nef from agroinfiltrated tissue was achieved by a two-step immobilized metal ion affinity chromatography protocol with yields of 250 ng/g of fresh tissue.</p> <p>Conclusion</p> <p>We demonstrated that expression level of HIV-1 Nef in plant can be improved using a transient expression system enhanced by the AMCV-P19 gene silencing suppressor protein. Moreover, plant-derived Nef was purified, with enhanced yield, exploiting a two-step purification protocol. These results represent a first step towards the development of a plant-derived HIV vaccine.</p

Plant antibodies for human antifungal therapy

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 β-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 β-glucan-binding specificity and the antifungal activities of the cognate murine mAb against C. albicans. In fact, they recognized preferentially β1,3-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

Human fibroblasts in vitro exposed to 2.45 GHz continuous and pulsed wave signals: Evaluation of biological effects with a multimethodological approach

The increasing exposure to radiofrequency electromagnetic fields (RF-EMF), especially from wireless communication devices, raises questions about their possible adverse health effects. So far, several in vitro studies evaluating RF-EMF genotoxic and cytotoxic non-thermal effects have reported contradictory results that could be mainly due to inadequate experimental design and lack of well-characterized exposure systems and conditions. Moreover, a topic poorly investigated is related to signal modulation induced by electromagnetic fields. The aim of this study was to perform an analysis of the potential non-thermal biological effects induced by 2.45 GHz exposures through a characterized exposure system and a multimethodological approach. Human fibroblasts were exposed to continuous (CW) and pulsed (PW) signals for 2 h in a wire patch cell-based exposure system at the specific absorption rate (SAR) of 0.7 W/kg. The evaluation of the potential biological effects was carried out through a multimethodological approach, including classical biological markers (genotoxic, cell cycle, and ultrastructural) and the evaluation of gene expression profile through the powerful high-throughput next generation sequencing (NGS) RNA sequencing (RNA-seq) approach. Our results suggest that 2.45 GHz radiofrequency fields did not induce significant biological effects at a cellular or molecular level for the evaluated exposure parameters and conditions

Designing greenhouse subsystems for a lunar mission: the LOOPS - M Project

The 2020s is a very important decade in the space sector, where international cooperation is moving towards the exploration of the Moon and will lead to stable lunar settlements, which will require new, innovative, and efficient technologies. In this context, the project LOOPS–M (Lunar Operative Outpost for the Production and Storage of Microgreens) was created by students from Sapienza University of Rome with the objective of designing some of the main features of a lunar greenhouse. The project was developed for the IGLUNA 2021 campaign, an interdisciplinary platform coordinated by Space Innovation as part of the ESA Lab@ initiative. The LOOPS-M mission was successfully concluded during the Virtual Field Campaign that took place in July 2021. This project is a follow-up of the V-GELM Project, which took part in IGLUNA 2020 with the realization in Virtual Reality of a Lunar Greenhouse: a simulation of the main operations connected to the cultivation module, the HORT3 , which was already developed by ENEA (Italian National Agency for New Technologies, Energy and Sustainable Economic Development) during the AMADEE-18 mission inside the HORTSPACE project. This paper will briefly describe the main features designed and developed for the lunar greenhouse and their simulation in a VR environment: an autonomous cultivation system able to handle the main cultivation tasks of the previous cultivation system, a bioconversion system that can recycle into new resources the cultivation waste with the use of insects as a biodegradation system, and a shield able of withstanding hypervelocity impacts and the harsh lunar environment. A wide overview of the main challenges faced, and lessons learned by the team to obtain these results, will be given. The first challenge was the initial inexperience that characterized all the team members, being for most the first experience with an activity structured as a space mission, starting with little to no know-how regarding the software and hardware needed for the project, and how to structure documentation and tasks, which was acquired throughout the year. An added difficulty was the nature of LOOPS-M, which included very different objectives that required different fields of expertise, ranging from various engineering sectors to biology and entomology. During the year, the team managed to learn how to handle all these hurdles and the organizational standpoint, working as a group, even if remotely due to the Covid-19 pandemic. Through careful planning, hard work and the help of supervisors, the activity was carried out through reviews, up to the prototyping phase and the test campaign with a successful outcome in each aspect of the project. By the end of the year everyone involved had acquired new knowledge, both practical and theoretical, and learned how to reach out and present their work to sponsors and to the scientific community

Designing a novel tomato ideotype for future cultivation in space manned missions

Introduction: Methods for production of fresh, health food are needed in view of long-term, deep-space manned missions. To this end, crops tailored for better performance under non-terrestrial conditions may be obtained by the exploitation of biochemical patterns related to specialized metabolites known to confer protection against environmental challenges and to be beneficial to human health.Methods: In this work, for the first time, MicroTom plants have been engineered specifically for agrospace applications to express PhAN4, a MYB-like transcription factor able to regulate the biosynthesis of anthocyanins that influence tomato genes possibly involved in agrospace-relevant functions. Results:PhAN4 engineering underpinned the genetic background of the dwarf tomato MicroTom while maintaining yield and photosynthetic capacity. PhAN4 expression resulted in the accumulation of anthocyanins and polyphenols, a differential carotenoid profile, increased antioxidant scavenging capacities of fruits compared to the original genotype. Improved ability to counteract ROS generation and to preserve plant protein folding after ex-vivo gamma irradiation was observed.Discussion: These results highlights that the manipulation of specific metabolic pathways is a promising approach to design novel candidate varieties for agrospace applications

Plants as biofactories: Production of pharmaceutical recombinant proteins

Abstract Plants can be used for the large-scale production of a variety of recombinant proteins, destined for agroindustrial, biomedical and pharmaceutical applications. In fact, blood components, hormones, enzymes, cytokines, antibodies and vaccines have been successfully produced in plants. Plant systems offer unique advantages in comparison with conventional techniques (bacteria, mammal or yeast cells): i) lower production costs; ii) synthesis of functional proteins, similar to those produced in animal cells, absolutely free of animal pathogens; iii) easy scale-up and purification technology. Furthermore, the engineering of edible plants may allow for the delivery of the recombinant protein (e.g. vaccines) through fruits, tubers, leaves or seeds. In this way the cold chain, necessary for the storage and the transport of purified recombinant products could be avoided, as well as the administration procedures by injection. This review describes gene transfer methods (including stable and transient transformation), plant species used and strategies to obtain high yields of protein, with attention focused towards plant-derived antibodies and vaccines, known as &quot;plantibodies&quot; or &quot;plantigens&quot;. Up to now, several groups are working in this promising field of research demonstrating that plants are able to produce proteins derived from different kingdoms, with highly complex structures (e.g. immunoglobulins A and G) and the different, post-translational, modification patterns of plants do not dramatically affect the properties or the biological activity of the recombinant protein. Using plants as biofactories to produce &quot;green&quot; therapeutic proteins is not only a proof of concept but a reality as an approach for agroindustry

Plant HSP70-induced multiepitope immune response

Summary 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