Performance of plant-produced RBDs as SARS-CoV-2 diagnostic reagents: a tale of two plant platforms.

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development.

Plant-Based Systems for Vaccine Production.

Plant systems have been used as biofactories to produce recombinant proteins since 1983. The huge amount of data, collected so far in this framework, suggests that plants display several key advantages over existing traditional platforms when they are intended for therapeutic uses, including safety, scalability, and the speed in obtaining the final product.Here, we describe a method that could be applied for the expression and production of a candidate subunit vaccine in Nicotiana benthamiana plants by transient expression, defining all the protocols starting from plant cultivation to target recombinant protein purification.

A Replicating Viral Vector Greatly Enhances Accumulation of Helical Virus-Like Particles in Plants.

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs.

Plant Virus Nanoparticles for Vaccine Applications.

In the rapidly evolving field of nanotechnology, plant virus nanoparticles (pVNPs) are emerging as powerful tools in diverse applications ranging from biomedicine to materials science. The proteinaceous structure of plant viruses allows the capsid structure to be modified by genetic engineering and/or chemical conjugation with nanoscale precision. This means that pVNPs can be engineered to display peptides and proteins on their external surface, including immunodominant peptides derived from pathogens allowing pVNPs to be used for active immunization. In this context, pVNPs are safer than VNPs derived from mammalian viruses because there is no risk of infection or reversion to pathogenicity. Furthermore, pVNPs can be produced rapidly and inexpensively in natural host plants or heterologous production platforms. In this review, we discuss the use of pVNPs for the delivery of peptide antigens to the host immune in pre-clinical studies with the final aim of promoting systemic immunity against the corresponding pathogens. Furthermore, we described the versatility of plant viruses, with innate immunostimulatory properties, in providing a huge natural resource of carriers that can be used to develop the next generation of sustainable vaccines.

Plant-Made Bet v 1 for Molecular Diagnosis.

Allergic disease diagnosis is currently experiencing a breakthrough due to the use of allergenic molecules in serum-based assays rather than allergen extracts in skin tests. The former methodology is considered a very innovative technology compared with the latter, since it is characterized by flexibility and adaptability to the patient's clinical history and to microtechnology, allowing multiplex analysis. Molecular-based analysis requires pure allergens to detect IgE sensitization, and a major goal, to maintain the diagnosis cost-effective, is to limit their production costs. In addition, for the production of recombinant eukaryotic proteins similar to natural ones, plant-based protein production is preferred to bacterial-based systems due to its ability to perform most of the post-translational modifications of eukaryotic molecules. In this framework, Plant Molecular Farming (PMF) may be useful, being a production platform able to produce complex recombinant proteins in short time-frames at low cost. As a proof of concept, PMF has been exploited for the production of Bet v 1a, a major allergen associated with birch (Betula verrucosa) pollen allergy. Bet v 1a has been produced using two different transient expression systems in Nicotiana benthamiana plants, purified and used in a new generation multiplex allergy diagnosis system, the patient-Friendly Allergen nano-BEad Array (FABER). Plant-made Bet v 1a is immunoreactive, binding IgE and inhibiting IgE-binding to the Escherichia coli expressed allergen currently available in the FABER test, thus suggesting an overall similar though non-overlapping immune activity compared with the E. coli expressed form.

Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes.

Plant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required.