Plant-made proteinaceous materials as biotechnological tool for diagnostic and therapeutic applications

Analytical techniques play a crucial role in science, offering rapid, sensitive, and precise reactions for detecting various issues, particularly those related to health. Over time, numerous analytical tools have been developed to characterize biomolecules, facilitate biosensing, diagnosis, and biomedical imaging. Examples include mass spectrometry, fluorescence spectroscopy, and techniques based on specific recognition events like enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), and radioimmunoassay (RIA). To provide for the specific needs of patients and enhance the effectiveness and safety of treatments, pharmaceutical companies are employing combinatory approaches that merge therapeutics and molecular diagnostics. These approaches aim to provide tailored clinical solutions, facilitate early disease diagnosis, and improve the quality of life for millions of individuals. Molecular diagnostics, which utilize molecular biology and analytic biochemistry, are widely employed procedures. They effectively identify specific biomarkers. Integrating relevant biomarkers as prediction tools in clinical trials is gaining traction as it enables smarter decisions, efficient resource investment, and the development of targeted treatments through companion diagnostics. While early research primarily focused on nucleic acid-based biomarkers such as DNA, SNPs, and mRNA expression patterns, current evidence suggests their potential utility as therapeutics and diagnostic tools may be limited. Protein biomarkers, on the other hand, offer a greater degree of differentiated information value and can help bridge this gap. Immunoassays remain the most sensitive, specific, and selective tool for examining such markers. In this context, the demand for recombinant proteins is growing in terms of quality, quantity, and diversity. This trend has attracted global attention towards the development, engineering, and utilization of new protein production technologies. Plant Molecular Farming (PMF), a biotechnology branch that harnesses plant genetic engineering, has proven to be a viable platform for manufacturing economically valuable, heterologous proteins. Furthermore, plant viruses have transformed from pathogens to molecular tools, facilitating recombinant protein expression, vaccine manufacturing, and serving as nano scaffolds for peptide display. This industrial Ph.D. project was conducted in Diamante Srl, a biotech company located in Verona. Diamante's research primarily focuses on autoimmune disease diagnosis and therapy, exploiting plants as bioreactors. Within this project, we present three examples of PMF combined with plant virus-based expression systems: 1. Development of in vitro diagnostic systems for autoimmune diseases using plant- made nanomaterials derived from plant viruses. 2. Production of a plant-based serological reagent for SARS-CoV-2 antibody tests. 3. Plant-based production and bacteriostatic evaluation of human tear lipocalin. The first application of PMF described here addresses the lack of a unified diagnostic system for autoimmune diseases such as Sjögren's Syndrome and Rheumatoid Arthritis. The prolonged diagnostic process and frequent misdiagnosis in these conditions have led to harmful consequences for patients. Our work focuses on the development and implementation of indirect ELISA tests for diagnosing these two autoimmune diseases. We achieve this by utilizing immunodominant peptides displayed on the surface of plant virus nanoparticles. The second focus stems from the outbreak of coronavirus disease caused by SARS-CoV-2. It has become evident that high-level recombinant protein expression is crucial for cost- effective production of viral proteins for various applications. Serological tests, for instance, serve as valuable tools to monitor humoral immunity resulting from infection or vaccination. In this project, our objective is to produce a plant-based SARS-CoV-2 Spike (S) Glycoprotein fragment, specifically the Receptor Binding Domain (RBD), and establish a serological test based on this antigen. We will then compare its performance with a commercial serological system. Lastly, we aim to evaluate the activity of plant-made human tear Lipocalin (TL) against four strains of hypervirulent Klebsiella Pneumoniae, a multidrug-resistant bacterium, in order to assess its potential bacteriostatic effects.