How Plant Molecular Farming Can Help Fight Against COVID‐19?
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Rahim Ghezzi,1,* Leila Nejadsadeghi,2
1. Shahid Chamran university
- Introduction: Abstract
Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is a new virus responsible for the COVID- 19 pandemic, which is the worst public health crisis of this Century. Urgent measures are needed to contain and control the virus, particularly diagnostic kits for detection and surveillance, therapeutics to reduce mortality among the severely affected, and vaccines to protect the remaining population. Some scientists working on plant biotechnology together with commercial enterprises for the
emergency manufacturing of diagnostics and therapeutics have aimed to fulfill the rapid demand for SARS‐CoV‐2 protein antigen and antibody through a rapid, scalable technology known as transient/stable expression in plants. Plant biotechnology using transient/stable expression offers a rapid solution to address this crisis through the production of low‐cost diagnostics, antiviral drugs, immunotherapy, and vaccines. Transient/stable expression technology for manufacturing plant based biopharmaceuticals is already established at commercial scale. Here we discuss the potential role of plant molecular farming in the rapid and scalable supply of protein antigens as reagents and vaccine candidates, antibodies for virus detection and opinions regarding how plan molecular farming can help fight against COVID‐19.
The COVID-19 pandemic is an urgent global health crisis in human history, which has dramatically affected the health, economy, and social mobility of almost everyone on the planet. The emergence of SARS-CoV-2 in late 2019 and its rapid spread in 2020 has posed several global challenges that demand new solutions in public healthcare and the biomedical research ecosystem (Webb et al., 2020). The normal life and livelihood of most world citizens have been disrupted, causing an incalculable economic
depression. Health officials and government agencies have imposed extreme measures to limit human mobility and social distancing strategies to slow down the infection rate, thus decreasing the total number of hospitalized patients at one time. This strategy also allows more time to find and develop effective testing reagents to identify carriers, find suitable antiviral drugs to treat severely affected patients, and develop a vaccine to protect the unexposed portion of the population. During this crisis, plant scientists can play a key role in developing new diagnostics reagents and therapeutics with their knowledge and plant‐based biopharmaceuticals infrastructure. In this article, we discuss how plant molecular farming
could provide practical solutions to address the outbreak of COVID-19. The World Health Organization (WHO) welcomes innovations around the world including repurposing drugs, traditional medicines and developing new therapies in the search for potential treatments for COVID-19. use of plants for the large-scale production of biopharmaceuticals (Molecular farming) represents an interesting and mature technology that has already proved its benefits in terms of safety, scalability, rapidity and reduced manufacturing costs. Monoclonal antibodies (mAbs) are useful tools in medicine, biology and biochemistry due to their binding specificity to different molecular targets and their stability both in vivo and in vitro. Mammalian cell cultures mainly based on Chinese Hamster Ovary cells (CHO) are still the favored system for the production of commercial mAbs, even if the increasing demand (also linked to the growing market) is promoting the development of alternative expression platforms. Indeed, the facilities necessary for large-scale production in mammalian cells require high initial investments and their operating and maintenance costs are high (Ecker et al., 2015). Among alternative expression systems, plants represent promising bioreactors for the large-scale production of recombinant proteins and antibodies. They offer an attractive expression platform with several advantages such as the absence of potential human pathogens, possibility to engineer a tailored antibody glycosylation profile, possibility to scale-up production by simply increasing the number of plants and competitiveness of manufacturing costs.
- Methods: Molecular Farming and the Plant-based Vaccines Technology
Post-translational modifications of proteins occurring in plant cells are essentially similar to those in animal cells and the correct assembly of complex molecules, such as antibodies, are assisted by chaperones that mediate the folding and formation of disulfide bonds while the addition of N-glycans is performed by specific cellular glycosyltransferases. Plant molecular farming encompasses a variety of
different expression technologies, ranging from stable nuclear transformation (transgenic plants) or plastid transformation (transplastomic plants) to transient expression without stable transgene integration (Fischer and Buyel 2020). Transient or epichromosomal transformation differs from stable transformation in that the exogenous sequence is not inherited by the progeny thus reducing the risk of environmental biosafety issues linked to the dissemination of the transgene through seeds or pollen. This approach generally provides high protein yields in a very short period of time (few days to weeks) which is not achievable via stable transformation (Komarova et al., 2010). Transient expression can be obtained using either vectors or plasmids containing T-DNA gene cassettes derived from A. tumefaciens bearing a strong constitutive promoter or vectors carrying appropriately modified genomic sequences of plant viruses inserted in T-DNA cassettes (viral vectors) (Kopertekh and Schiemann 2017). Given the urgent need for diagnostics, vaccines, and therapeutics for a rapidly-spreading novel or re-emerging disease, only transient expression systems provide the necessary speed and scalability (Tusé et al., 2020).
Currently, Agrobacterium-mediated transformation is the most popular method to achieve this modification since this bacterium has the ability to transfer large segments of DNA with minimal rearrangement at high efficiency with low number of insertions. Nonetheless, the transgene is randomly inserted into the genome, which often leads to positional effects that make expression levels unpredictable and interruption of endogenous genes a possibility. Another limitation is the induction of silencing mechanisms that hamper productivity. Nevertheless, it should be considered that new technologies are emerging to cope with these limitations by providing ways to achieve site-directed insertion through a number of mechanisms.
Table 1: Description of the expression approaches for the production of plant-based vaccines and precedents for MERS/SARS-CoV-1 vaccines.
Approach Attractive Features Drawbacks Proposed Target Antigens MERS/SARS Precedents Reference
Stable nuclear genome transformation Inheritable antigen production, allows seed bank generation;
post-translational modifications are performed;
protocols available for several species including seed crops Non-site specific transgene insertion;
horizontal gene transfer is possible;
transgene expression affected by position effects and silencing;
transformation takes long time S protein;
multiepitope vaccines The N-terminal fragment of the SARS-CoV-1 S protein (S1) was expressed in stably transformed tomato and low-nicotine tobacco plants, which induced IgA and IgG responses in mice. (Pogrebnyak et al., 2005)
Transient nuclear genome transformation Rapid production;
high productivity;
implemented at the industrial level Seed bank cannot be generated;
requires purification of the antigen to eliminate toxic compounds from the host and ag-robacteria residues S protein;
multiepitope vaccines A chimeric protein of GFP and amino acids 1-658 of the SARS-CoV-1 S protein (S1:GFP) was transiently expressed in tobacco leaves and stably transformed in tobacco and lettuce. No immunization assays were performed
The SARS-CoV-1 N protein was transiently expressed in Nicotiana benthamiana, which induced in mice high levels of IgG1 and IgG2a and up regulation of IFN-γ and IL-10 in splenocytes.
A chimeric protein of GFP and the SARS-CoV-1 S protein was transiently expressed in tobacco plants. No immunization tests were performed.
The SARS-CoV-1 M and N proteins were transiently expressed in N. benthamiana. The N protein was antigenic but immunogenicity was not assessed. (Li et al., 2006; Zheng et al., 2009; Demurtas et al., 2016)
Using transient expression systems, plants may offer the only platform that can be used to produce diagnostics and therapeutics at a large scale in a few weeks, which is extremely relevant to the current pandemic situation, and they can be scaled up rapidly to address unforeseen and sudden demands.
Possibilities to Develop Anti-COVID-19 Plant-based Vaccines
In the last 20 years, plants have become vital competitors to bacteria, yeast, and mammalian cell‐based production systems for biopharmaceuticals. Plants are highly efficient in producing proteins of varying complexity, serving as a bioreactor/mini factory for manufacturing protein‐based diagnostics and therapeutics. Plant‐based biopharmaceutical production platforms exhibit agility, accuracy, and speed
by eliminating the risk of mutation and contamination during production and significantly shortening production timelines. Plants have much to offer for fighting against COVID‐19.
1. Diagnostic reagents
The effective management of COVID-19 requires an increase in diagnostic capacity, particularly the development, manufacture, and stockpiling of assays to detect the SARS-CoV-2 genome and/or antigens itself or the antibodies it elicits. Accurate antibody tests for COVID-19 require high-quality reagents, although differences between analytical and clinical sensitivity has not yet been defined for any test. The huge demand for diagnostic kits has highlighted not only the critical shortage of reagents (recombinant antigens and antibodies) but also the means to produce them. Plants have already been shown to produce SARS-CoV antigens (Demurtas et al., 2016). The nucleoprotein (N), transiently expressed in N. benthamiana, was recognized by sera from Chinese SARS-convalescent patients around the time of the 2003 outbreak. Furthermore, the full-length membrane (M) protein was produced in plants but not in bacteria due to unanticipated toxicity (Carattoli et al., 2005). This provided proof of principle that plants could be used as a robust, rapid and flexible production system for SARS diagnostic reagents, potentially allowing the development of immunological assays for stockpiling in case of recurring SARS outbreaks (De Martinis et al., 2016).
Due to its rapid spreading nature, the COVID‐19 pandemic has created a sudden huge crisis for diagnostics, and consequently caused severe shortage in the diagnostic reagents and materials to manufacture them. Currently, two types of diagnostics are in high demand. The first one is the antigen test to detect the virus directly and thus identify, separate, and treat the infected populations. The second one is the antibody test to detect the antibody produced against the viral infection and thus identify the infected, convalescent, and immune populations. There are two types of antigen tests: the first one based on the detection of viral genomic RNA, and the second based on the detection of viral proteins. In the RNA‐based test, the virus is detected by quantitative reverse transcription PCR (RT‐qPCR), for which we only need to synthesize gene‐specific primers from the published genome sequence of SARS‐CoV‐2. However, a major problem with the RT‐PCR‐based test is the lack of a universal positive control; thus, there is a possibility of false positive or false negative results. This problem can be solved by developing plantderived virus‐like particles (VLPs) as a universal positive control for the RT‐qPCR test. (Figure 1)
The glycosylation patterns of proteins that form VLPs can impact their immunogenicity and protective capacity. Interestingly, glycoengineering strategies have been successfully implemented for plants, which allows diversifying their application as hosts for the production of biopharmaceuticals. This is a relevant aspect considering that plants lack the ability to perform glycosylation, which is a characteristic of mammalian systems. For instance, plants perform beta1,2-xylosylation, core alpha1,3-fucosylation, and the addition of a second N-acetylglucosamine (GlcNAc) to the mannose core. Moreover, plant glycans lack β (1,4)-galactose and sialic acid, as well as bi-antennary N-glycans (Lerouge et al., 2009).
Virus‐like particles (VLPs) based on plant viruses represent an exciting prospect for vaccine development. VLPs mimic the original structure of virus, which allows them to be easily recognized by the immune system of the host. VLPs lack core genetic materials, which ensures an extra layer of safety, as they cannot replicate in humans, making them non‐infectious, and can be manufactured in huge quantities by transient expression in plants (Rybicki E. P. 2020)
Figure 1. Developmental routes for plant‐based diagnostics and therapeutics to address the COVID‐19 crisis. Blue arrows indicate transient expression; brown arrow indicates stable expression; olive green arrows indicate potential routes for diagnostics; and light green arrows indicate potential routes for therapeutics manufacturing platforms. SARS‐CoV‐2: severe acute respiratory syndrome coronavirus 2; VLPs: virus‐like particles. Some of these images were generated using Biorender (https://biorender.com/).
Although plant-derived SARS-CoV-2 VLPs have yet to be reported, the feasibility of this approach has been demonstrated by the successful production of other coronavirus VLPs in insect and mammalian cells (Lu et al., 2007; Bai et al., 2008; Lokugamage et al., 2008). This suggests that SARS-CoV2 VLPs could be assembled in plants by co-expressing the M, E, and S proteins. Medicago announced a program to develop a VLPbased COVID-19 vaccine candidate in July 2020, combining their recombinant coronavirus virus-like particle (CoVLP) technology with adjuvants from GlaxoSmithKline and Dynavax
Technologies for the phase I trial8. A VLP-based COVID-19 vaccine program has also been announced by iBio Inc.6 This company was established with funding from the United States Defense Advanced Research Projects Agency (DARPA) and was part of the Blue Angel initiative to establish centers for the rapid delivery of medical countermeasures in response to emerging diseases, as demonstrated by the production of 10 million doses of influenza vaccine in only 1 month using its plant-based9.
2. Vaccine
Vaccines are the most economical and effective way to control and prevent any infectious disease. Therefore, the development of an appropriate vaccine against COVID‐ 19 is urgent. Recent investigations have demonstrated that SARS‐CoV‐2 structural proteins can stimulate neutralizing antibodies and enhance the CD4+/CD8+ T cell response (Shang et al., 2020). SARS‐CoV‐2 consists of four structural proteins. Among them, the N protein is highly conserved in the CoV family, whereas the M and E proteins induce a weak protective response (Figure 2), indicating that the N, M, and E proteins are unsuitable for targets vaccine candidates (Gralinski, and Menachery 2020). Therefore, the S protein is the main target for vaccine candidates. However, the S protein of SARS‐CoV‐2 is divided into the S1 and S2 subunits. S2 is membrane‐spanning and highly conserved (99%) in the CoV family, whereas the S1 subunit shows only 70% individuality to other strains of human corona virus. Blocking viral entry to the host cells is a promising strategy to control infection, and most of the vaccines for the SARSCoV have targeted the S1 subunit for this reason (Du et al., 2009). Manufacturing subunit vaccines based on individual proteins, producing either virus subunit antigens or VLPs, is a safer and quicker alternative than vaccines developed through conventional approaches using inactivated or attenuated strains.
Figure 2. Structure of the SARS-CoV-2 virus. The virus is formed by an envelope membrane associated with the following structural proteins: spike protein (S), which mediates binding to the host cell receptors and considered a critical target for the induction of antibodies capable of neutralizing the virus; hemagglutinin-esterase dimer (HE), which acts as a potent mediator of attachment and destruction of sialic acid receptors on the host cell surface; a membrane glycoprotein (M), which is important to generate the virus; and the envelope protein (E), which adheres to the M protein to form the viral envelope. The viral structure also comprises a nucleocapsid protein (N) that, along with the RNA genome, produces the nucleocapsid.
Many subunit vaccine candidates for pandemic or seasonal strains of influenza have already been developed by transient expression in the tobacco plant. Vaccine antigens were produced with a deconstructed vector based on the tobacco mosaic virus delivered by the agroinfiltration technique with Agrobacterium tumefaciens. This technology ensures uniformly high levels of target protein expression in Nicotiana benthamiana and can produce a maximum of 200 mg of protein per kg of tobacco leaves within 3 weeks just after receiving the corresponding sequences. From the CoV family, only one previous report demonstrated that the S1 subunit of swine‐transmissible gastroenteritis coronavirus (TGEV) expressed in Arabidopsis thaliana transgenic lines produces recombinant antigen‐elicited TGEV‐specific antibodies in mice, indicating that immunogenic CoV antigens can be expressed and produced in plants (Gomez et al., 1998).
The S protein of several avian, swine and murine coronaviruses, as well as the N-terminal fragment of the
SARS-CoV S protein, have been produced successfully in transgenic maize, potato, tomato, or tobacco plants by classic Agrobacterium-mediated transformation, or by display on the surface of plant viruses, and in all cases the products induced an immune response following oral delivery (Tuboly et al., 2000;
Bae et al., 2003; Lamphear et al., 2004; Zhou et al., 2004) or nasal delivery (Koo et al., 1999). However, transient expression is more suitable for the speed and scale of production needed to address a rapidly-spreading disease live COVID-19.
- Results: Result
- Conclusion: Conclusions
The emergence of COVID-19 has led to a global emergency that demands the development of new biologics, especially vaccines, to counteract against this threat. In this scenario, a plant-made vaccine is a viable approach to rapidly respond to this need. The current expression technologies offer relevant paths for developing anti-COVID-19 vaccines. Plant Molecular Farming through transient/stable expression in plants offers an outstanding platform to produce biopharmaceuticals to fight against COVID‐19. Transient/stable expression in plants is a faster, cost effective, scalable, and flexible technology than traditional microbe, insect, or mammalian cell‐based platforms because there is no need to establish stable culture cell lines or costly culture media, neither is there any need for an extra set‐up for the scaled‐up production except for the cultivation of more plants. Crop plants can be grown in diverse environments; therefore, biopharmaceuticals could be produced using already established infrastructures for agricultural production and the same distribution networks that exist for the supply of food and cereal seeds, without the need for a cold supply chain. Plant Molecular Farming has the opportunity not only to fight against COVID‐19 but also to create a perfect model that allows a rapid and intended response to any crises in the future.
- Keywords: COVID-19, Plant molecular farming, recombinant proteins, transient expression
