Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells

Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts, Bio-Encapsulation, Stability and Functional Evaluation In Vitro

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts

Improving the active involvement of stakeholders and the public in flood risk management – tools of an involvement strategy and case study results from Austria, Germany and Italy

The EU Flood Risk Management Directive 2007/60/EC aims at an active involvement of interested parties in the setting up of flood risk management plans and thus calls for more governance-related decision-making. This requirement has two perspectives. On the one hand, there is (1) the question of how decision-makers can improve the quality of their governance process. On the other hand, there is (2) the question of how the public shall be appropriately informed and involved. These questions were the centre of the ERA-Net CRUE-funded project IMRA (integrative flood risk governance approach for improvement of risk awareness) that aimed at an optimisation of the flood risk management process by increasing procedural efficiency with an explicit involvement strategy. To reach this goal, the IMRA project partners developed two new approaches that were implemented in three case study areas for the first time in flood risk management: 1. risk governance assessment tool: An indicator-based benchmarking and monitoring tool was used to evaluate the performance of a flood risk management system in regard to ideal risk governance principles; 2. social milieu approach: The concept of social milieus was used to gain a picture of the people living in the case study regions to learn more about their lifestyles, attitudes and values and to use this knowledge to plan custom-made information and participation activities for the broad public. This paper presents basic elements and the application of two innovative approaches as a part of an "involvement strategy" that aims at the active involvement of all interested parties (stakeholders) for assessing, reviewing and updating flood risk management plans, as formulated in the EU Flood Risk Management Directive 2007/60/EC