Vaccine production using plants as alternative expression system

The COVID-19 pandemic and the increasing frequency of global health emergencies have emphasized the need for vaccine manufacturing platforms that are not only rapid and flexible, but also scalable, sustainable, and globally accessible. Conventional expression systems based on mammalian or microbial cells are constrained by high production costs, complex infrastructure, and limited scalability in low-resource settings. In this context, Plant Molecular Farming (PMF) has emerged as a compelling alternative, leveraging the ability of plants to produce complex biopharmaceuticals. PMF offers short production times, low capital investment, and the potential for decentralized biomanufacturing, thus addressing critical limitations of traditional vaccine supply chains. This PhD research explored PMF as an alternative platform for vaccine production, with a particular focus on downstream processing and purification, the key bottlenecks that remain against industrial translation. Two complementary vaccine strategies were investigated: recombinant protein vaccines and nucleic acid–based vaccines, enabling a comparative assessment of process feasibility and analytical challenges. The project was structured into two complementary phases, one conducted in an industrial setting and the other in an academic environment, allowing to investigate both perspectives. The first phase was carried out at Special Product’s Line (SPL, Anagni, Italy) and addressed the development of a downstream process for TB002, a recombinant subunit vaccine candidate against tuberculosis produced in Nicotiana benthamiana. Process optimization included buffer screening, immobilized metal affinity chromatography, thrombin-mediated tag removal, and final polishing. Despite challenges such as aggregation and protein loss during sterilization, a robust protocol was established, enabling the expression of a recombinant subunit vaccine suitable for preclinical validation. The second phase was conducted at University College London (UCL, London, UK) and focused on nucleic acid–based vaccine platforms. A model mRNA vaccine encoding GFP was adapted for plant expression using the pGreenII binary vector and delivered into N. benthamiana via Agrobacterium-mediated infiltration. Expression was confirmed in planta, and a range of extraction and purification methods were compared, including anion-exchange and oligo(dT) chromatography, magnetic beads capture, and commercial silica kits. While transcript recovery was achieved, yields and purity were strongly method-dependent, highlighting the technical complexity of plant-derived mRNA purification. In parallel, virus-like particles (VLPs) derived from Cowpea Chlorotic Mottle Virus (CCMV) were developed as protective nanocapsule for mRNA. The coat protein 7 was expressed in both E. coli and N. benthamiana, purified using a combination of chromatographic strategies, and tested for assembly/disassembly under pH-controlled conditions. These studies provided proof-of-concept for plant-based VLP production and their potential application in mRNA encapsulation. Overall, this PhD work demonstrates both the opportunities and bottlenecks of Plant Molecular Farming for producing vaccines. Industrial- scale efforts highlight the feasibility of GMP-compatible processes, while academic investigations expand the scope of PMF toward next-generation platforms integrating mRNA production and VLP-mediated delivery. Together, these findings support the potential of plants as versatile, sustainable, and scalable biofactories for future vaccine development.