Pilot Production of SARS-CoV-2 Related Proteins in Plants: A Proof of Concept for Rapid Repurposing of Indoor Farms Into Biomanufacturing Facilities

Abstract

The current CoVid-19 crisis is revealing the strengths and the weaknesses of the world’s capacity to respond to a global health crisis. A critical weakness has resulted from the excessive centralization of the current biomanufacturing capacities, a matter of great concern, if not a source of nationalistic tensions. On the positive side, scientific data and information have been shared at an unprecedented speed fuelled by the preprint phenomena, and this has considerably strengthened our ability to develop new technology-based solutions. In this work, we explore how, in a context of rapid exchange of scientific information, plant biofactories can serve as a rapid and easily adaptable solution for local manufacturing of bioreagents, more specifically recombinant antibodies. For this purpose, we tested our ability to produce, in the framework of an academic lab and in a matter of weeks, milligram amounts of six different recombinant monoclonal antibodies against SARS-CoV-2 in Nicotiana benthamiana. For the design of the antibodies, we took advantage, among other data sources, of the DNA sequence information made rapidly available by other groups in preprint publications. mAbs were engineered as single-chain fragments fused to a human gamma Fc and transiently expressed using a viral vector. In parallel, we also produced the recombinant SARS-CoV-2 N protein and the receptor binding domain (RBD) of the Spike protein in planta and used them to test the binding specificity of the recombinant mAbs. Finally, for two of the antibodies, we assayed a simple scale-up production protocol based on the extraction of apoplastic fluid. Our results indicate that gram amounts of anti-SARS-CoV-2 antibodies could be easily produced in little more than 6 weeks in repurposed greenhouses with little infrastructure requirements using N. benthamiana as production platform. Similar procedures could be easily deployed to produce diagnostic reagents and, eventually, could be adapted for rapid therapeutic responses.

Plant-Produced Receptor-Binding Domain of SARS-CoV-2 Elicits Potent Neutralizing Responses in Mice and Non-human Primates

“…Several plant-produced therapeutics ( Kizhner et al, 2015 ; Ma et al, 2015 ; Mor, 2015 ; Prevail Ii Writing Group et al, 2016 ) and vaccines ( Yuki et al, 2013 ; Hendin et al, 2017 ; Donini and Marusic, 2019 ) are currently in pipeline for clinical trials and approval, Notably plant-produced Glucocerebrosidase enzyme (Elelyso TM ) for the treatment of type I Gaucher’s disease has been approved by US Food and Drug Administration ( Fox, 2012 ) and tobacco-derived seasonal flu VLP vaccine from Medicago Inc., is currently in final stages of clinical trial ( Ward et al, 2021 ). Hence, plant-based expression could be an alternative option for rapid production of emergency vaccines or therapeutic antibodies ( Iyappan et al, 2018 ; Diego-Martin et al, 2020 ; Rattanapisit et al, 2020 ; Shanmugaraj and Phoolcharoen, 2021 ; Siriwattananon et al, 2021 ).…”

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

“…We expressed an RBD variant that was previously produced in mammalian or insect cells ( Stadlbauer et al, 2020 ; Klausberger et al, 2021 ) and observed low expression levels and high amounts of homodimers. Recombinant SARS-CoV-2 RBD variants have recently been produced in N. benthamiana using different transient expression systems ( Diego-Martin et al, 2020 ; Mamedov et al, 2020 ; Rattanapisit et al, 2020 ; Makatsa et al, 2021 ). Diego-Martin et al (2020) used a MagnICON-based expression vector to produce a His-tagged RBD variant carrying the same amino acid region (R319-F541) as present in our RBD.…”

“…Recombinant SARS-CoV-2 RBD variants have recently been produced in N. benthamiana using different transient expression systems ( Diego-Martin et al, 2020 ; Mamedov et al, 2020 ; Rattanapisit et al, 2020 ; Makatsa et al, 2021 ). Diego-Martin et al (2020) used a MagnICON-based expression vector to produce a His-tagged RBD variant carrying the same amino acid region (R319-F541) as present in our RBD. In agreement with our data, low yields (2-4 μg/g fresh weight) and considerable amounts of higher molecular weight bands were reported.…”

“…Despite these efforts, recombinant viral glycoproteins often accumulate at low levels in plants ( Margolin et al, 2018 ). They are poorly processed ( Le Mauff et al, 2015 ) and their expression can be associated with a stress response in the host leading to massive tissue necrosis ( Phoolcharoen et al, 2011 ; Diego-Martin et al, 2020 ). The expression of the HIV-1 Env glycoprotein in N. benthamiana leaves, for instance, resulted in a severe phenotype and activation of the unfolded protein response (UPR), suggesting that the host biosynthetic machinery does not support efficient production of the protein ( Margolin et al, 2019 , 2020a ).…”

The C-Terminal Half of SARS-CoV-2 Nucleocapsid Protein, Industrially Produced in Plants, Is Valid as Antigen in COVID-19 Serological Tests

“…The favorable features of this industrial approach have been widely and frequently reviewed, among which speed of production in very large amounts and comparative low costs of productions stand out (Ma et al, 2003;Williams et al, 2014Williams et al, , 2016Moon et al, 2019;Capell et al, 2020;McDonald and Holtz, 2020). In the particular case of SARS-CoV-2 proteins, a few examples showing their production in plants have been published very recently (Diego-Martin et al, 2020;Siriwattananon et al, 2021). A previous approach to produce plant-made viral proteins that could be potentially used as antigens has been shown for the related SARS-CoV, where antibody binding to immobilized plant-produced virus antigen was demonstrated using a limited panel of serum samples (Demurtas et al, 2016).…”