ABSTRACT
This manuscript describes a unique protocol for the rapid transformation of Medicago truncatula A17 cell suspension cultures mediated by Agrobacterium tumefaciens. Medicago cells were collected on day 7 of the growth curve, which corresponded to the beginning of the exponential phase. They were then co-cultured with Agrobacterium for 3 days before being spread onto a petri dish with appropriate antibiotic selection. The Receptor Binding Domain of the Spike protein of SARS-CoV-2 was used as a model to develop this protocol. The presence of the transgene was assessed using PCR, and the integrity of the product was evaluated by SDS-PAGE and Western-blotting.
ABSTRACT
The COVID-19 pandemic, caused by the worldwide spread of SARS-CoV-2, has prompted the scientific community to rapidly develop efficient and specific diagnostics and therapeutics. A number of avenues have been explored, including the manufacture of COVID-related proteins to be used as reagents for diagnostics or treatment. The production of RBD and Spike proteins was previously achieved in eukaryotic cells, mainly mammalian cell cultures, while the production in microbial systems has been unsuccessful until now. Here we report the effective production of SARS-CoV-2 proteins in two plant model systems. We established transgenic tobacco BY-2 and Medicago truncatula A17 cell suspension cultures stably producing the full-length Spike and RBD recombinant proteins. For both proteins, various glycoforms were obtained, with higher yields in Medicago cultures than BY-2. This work highlights that RBD and Spike can be secreted into the culture medium, which will impact subsequent purification and downstream processing costs. Analysis of the culture media indicated the presence of the high molecular weight Spike protein of SARS-CoV-2. Although the production yields still need improvement to compete with mammalian systems, this is the first report showing that plant cell suspension cultures are able to produce the high molecular weight Spike protein. This finding strengthens the potential of plant cell cultures as production platforms for large complex proteins.
ABSTRACT
The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.
Subject(s)
Artemisia annua , Communicable Diseases , Pharmaceutical Preparations , Animals , Humans , Molecular Farming , Plants, EdibleABSTRACT
Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause Ë17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.