Putting the spotlight back on plant suspension cultures

Plant cell suspension cultures have several advantages that make them suitable for the production of recombinant proteins. They can be cultivated under aseptic conditions using classical fermentation technology, they are easy to scale-up for manufacturing, and the regulatory requirements are similar to those established for well-characterized production systems based on microbial and mammalian cells. It is therefore no surprise that taliglucerase alfa (Elelyso®) – the first licensed recombinant pharmaceutical protein derived from plants – is produced in plant cell suspension cultures. But despite this breakthrough, plant cells are still largely neglected compared to transgenic plants and the more recent plant-based transient expression systems. Here, we revisit plant cell suspension cultures and highlight recent developments in the field that show how the rise of plants cells parallels that of Chinese hamster ovary cells, currently the most widespread and successful manufacturing platform for biologics. These developments include medium optimization, process engineering, statistical experimental designs, scale-up/scale-down models and process analytical technologies. Significant yield increases for diverse target proteins will encourage a gold rush to adopt plant cells as a platform technology, and the first indications of this breakthrough are already on the horizon

Examination of clinical and environmental Vibrio parahaemolyticus isolates by multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA)

Vibrio parahaemolyticus is a leading cause of seafood-borne infections in the US. This organism has a high genetic diversity that complicates identification of strain relatedness and epidemiological investigations. However, sequence-based analysis methods are promising tools for these identifications. In this study, Multi-Locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) was performed on 58 V. parahaemolyticus isolates (28 of oyster and 30 of clinical origin), to identify differences in phylogeny. The results obtained by both methods were compared to Pulsed-Field Gel Electrophoresis (PFGE) patterns determined in a previous study. Forty-one unique sequence types (STs) were identified by MLST among the 58 isolates. Almost half of the isolates (22) belonged to a new ST and added to the MLST database. A ST could not be generated for 5 (8.6%) isolates, primarily due to an untypable recA locus. Analysis with eBURST did not identify any clonal complex among the strains analyzed and revealed 37 singeltons with 4 of them forming 2 groups (1 of them SLV, and the other a DLV). An established MLVA assay, targeting 12 total genes through three separate 4-plex PCRs, was successfully adapted to high resolution melt (HRM) analysis with faster and easier experimental setup; resulting in 58 unique melt curve patterns. HRM-MLVA was capable of differentiating isolates within the same PFGE cluster and having the same ST. Conclusively, combining the three methods PFGE, MLST, and HRM-MLVA, for the phylogenetic analysis of V. parahaemolyticus resulted in a high resolution subtyping scheme for V. parahaemolyticus. This scheme will be useful as a phylogenetic research tool and as an improved method for outbreak investigations for V. parahaemolyticus

Application of a scalable plant transient gene expression platform for malaria vaccine development

Despite decades of intensive research efforts there is currently no vaccine that provides sustained sterile immunity against malaria. In this context, a large number of targets from the different stages of the Plasmodium falciparum life cycle have been evaluated as vaccine candidates. None of these candidates has fulfilled expectations, and as long as we lack a single target that induces strain-transcending protective immune responses, combining key antigens from different life cycle stages seems to be the most promising route towards the development of efficacious malaria vaccines. After the identification of potential targets using approaches such as omics-based technology and reverse immunology, the rapid expression, purification and characterization of these proteins, as well as the generation and analysis of fusion constructs combining different promising antigens or antigen domains before committing to expensive and time consuming clinical development, represents one of the bottlenecks in the vaccine development pipeline. The production of recombinant proteins by transient gene expression in plants is a robust and versatile alternative to cell-based microbial and eukaryotic production platforms. The transfection of plant tissues and/or whole plants using Agrobacterium tumefaciens offers a low technical entry barrier, low costs and a high degree of flexibility embedded within a rapid and scalable workflow. Recombinant proteins can easily be targeted to different subcellular compartments according to their physicochemical requirements, including post-translational modifications, to ensure optimal yields of high quality product, and to support simple and economical downstream processing. Here we demonstrate the use of a plant transient expression platform based on transfection with A. tumefaciens as essential component of a malaria vaccine development workflow involving screens for expression, solubility and stability using fluorescent fusion proteins. Our results have been implemented for the evidence-based iterative design and expression of vaccine candidates combining suitable P. falciparum antigen domains. The antigens were also produced, purified and characterized in further studies by taking advantage of the scalability of this platform

Herbaceous plant species invading natural areas tend to have stronger adaptive root foraging than other naturalized species

Although plastic root-foraging responses are thought to be adaptive, as they may optimize nutrient capture of plants, this has rarely been tested. We investigated whether nutrient-foraging responses are adaptive, and whether they pre-adapt alien species to become natural-area invaders. We grew 12 pairs of congeneric species (i.e., 24 species) native to Europe in heterogeneous and homogeneous nutrient environments, and compared their foraging responses and performance. One species in each pair is a USA natural-area invader, and the other one is not. Within species, individuals with strong foraging responses, measured as plasticity in root diameter and specific root length, had a higher biomass. Among species, the ones with strong foraging responses, measured as plasticity in root length and root biomass, had a higher biomass. Our results therefore suggest that root foraging is an adaptive trait. Invasive species showed significantly stronger root-foraging responses than non-invasive species when measured as root diameter. Biomass accumulation was decreased in the heterogeneous vs. the homogeneous environment. In aboveground, but not belowground and total biomass, this decrease was smaller in invasive than in non-invasive species. Our results show that strong plastic root-foraging responses are adaptive, and suggest that it might aid in pre-adapting species to becoming natural-area invaders