Green Biologics: Harnessing the Power of Plants to Produce Pharmaceuticals.

Plants are increasingly used for the production of high-quality biological molecules for use as pharmaceuticals and biomaterials in industry. Plants have proved that they can produce life-saving therapeutic proteins (Elelyso™-Gaucher's disease treatment, ZMapp™-anti-Ebola monoclonal antibodies, seasonal flu vaccine, Covifenz™-SARS-CoV-2 virus-like particle vaccine); however, some of these therapeutic proteins are difficult to bring to market, which leads to serious difficulties for the manufacturing companies. The closure of one of the leading companies in the sector (the Canadian biotech company Medicago Inc., producer of Covifenz) as a result of the withdrawal of investments from the parent company has led to the serious question: What is hindering the exploitation of plant-made biologics to improve health outcomes? Exploring the vast potential of plants as biological factories, this review provides an updated perspective on plant-derived biologics (PDB). A key focus is placed on the advancements in plant-based expression systems and highlighting cutting-edge technologies that streamline the production of complex protein-based biologics. The versatility of plant-derived biologics across diverse fields, such as human and animal health, industry, and agriculture, is emphasized. This review also meticulously examines regulatory considerations specific to plant-derived biologics, shedding light on the disparities faced compared to biologics produced in other systems.

Plant-based expression and characterization of SARS-CoV-2 virus-like particles presenting a native spike protein.

We have investigated the use of transient expression to produce virus-like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID-19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full-length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic 'crown-shaped' or 'spiky' structure could be purified by density gradient centrifugation. Enzyme-linked immunosorbent assays using anti-S antibodies showed that threefold higher levels of VLPs containing the full-length S protein were obtained by infiltration with S alone, compared to co-infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID-19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant-made material is noninfectious, it does not have to be handled under conditions of high containment.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

Plant-made dengue virus-like particles produced by co-expression of structural and non-structural proteins induce a humoral immune response in mice.

Dengue virus (DENV) is an emerging threat causing an estimated 390 million infections per year. Dengvaxia, the only licensed vaccine, may not be adequately safe in young and seronegative patients; hence, development of a safer, more effective vaccine is of great public health interest. Virus-like particles (VLPs) are a safe and very efficient vaccine strategy, and DENV VLPs have been produced in various expression systems. Here, we describe the production of DENV VLPs in Nicotiana benthamiana using transient expression. The co-expression of DENV structural proteins (SP) and a truncated version of the non-structural proteins (NSPs), lacking NS5 that contains the RNA-dependent RNA polymerase, led to the assembly of DENV VLPs in plants. These VLPs were comparable in appearance and size to VLPs produced in mammalian cells. Contrary to data from other expression systems, expression of the protein complex prM-E was not successful, and strategies used in other expression systems to improve the VLP yield did not result in increased yields in plants but, rather, increased purification difficulties. Immunogenicity assays in BALB/c mice revealed that plant-made DENV1-SP + NSP VLPs led to a higher antibody response in mice compared with DENV-E domain III displayed inside bluetongue virus core-like particles and a DENV-E domain III subunit. These results are consistent with the idea that VLPs could be the optimal approach to creating candidate vaccines against enveloped viruses.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

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 ˜17% 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.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

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.

Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy.

The side effects of chemotherapy can be reduced by targeting tumor cells with an enzyme (or the corresponding gene) that converts a nontoxic prodrug into a toxic drug inside the tumor cells, also killing the surrounding tumor cells via the bystander effect. Viruses are the most efficient gene delivery vehicles because they have evolved to transfer their own nucleic acids into cells, but their efficiency must be balanced against the risks of infection, the immunogenicity of nucleic acids, and the potential for genomic integration. We therefore tested the effectiveness of genome-free virus-like particles (VLPs) for the delivery of Herpes simplex virus 1 thymidine kinase (HSV1-TK), the most common enzyme used in prodrug conversion therapy. HSV1-TK is typically delivered as a gene, but in the context of VLPs, it must be delivered as a protein. We constructed VLPs and smaller core-like particles (CLPs) based on Bluetongue virus, with HSV1-TK fused to the inner capsid protein VP3. TK-CLPs and TK-VLPs could be produced in large quantities in plants. The TK-VLPs killed human glioblastoma cells efficiently in the presence of ganciclovir, with an IC50 value of 14.8 µM. Conversely, CLPs were ineffective because they remained trapped in the endosomal compartment, in common with many synthetic nanoparticles. VLPs are advantageous because they can escape from endosomes and therefore allow HSV1-TK to access the cytosolic adenosine triphosphate (ATP) required for the phosphorylation of ganciclovir. The VLP delivery strategy of TK protein therefore offers a promising new modality for the treatment of cancer with systemic prodrugs such as ganciclovir.

Encapsidation of Viral RNA in Picornavirales: Studies on Cowpea Mosaic Virus Demonstrate Dependence on Viral Replication.

To elucidate the linkage between replication and encapsidation in Picornavirales, we have taken advantage of the bipartite nature of a plant-infecting member of this order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (empty virus-like particles [eVLPs]) can be generated by transiently coexpressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24,000-molecular-weight (24K) protease, in the absence of the replication machinery (K. Saunders, F. Sainsbury, and G. P. Lomonossoff, Virology 393:329-337, 2009, https://doi.org/10.1016/j.virol.2009.08.023). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in the Picornavirales We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effects of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication.IMPORTANCE The mechanism whereby members of the order Picornavirales specifically package their genomic RNAs is poorly understood. Research with monopartite members of the order, such as poliovirus, indicated that packaging is linked to replication, although the presence of "packaging signals" along the length of the viral RNA has also been suggested. Thanks to the bipartite nature of the CPMV genome, which allows the manipulation of RNA-1 without modifying RNA-2, we show here that this specificity is due to a functional link between the two processes of viral replication and encapsidation. This has important implications for our understanding of the fundamental molecular biology of Picornavirales and opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.

Investigating the Biological Relevance of In Vitro-Identified Putative Packaging Signals at the 5' Terminus of Satellite Tobacco Necrosis Virus 1 Genomic RNA.

Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that >90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only <1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA.IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite tobacco necrosis virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules.

Amino acids at the exposed C-terminus of the S coat protein of cowpea mosaic virus play different roles in particle formation and viral systemic movement.

The icosahedral capsid of cowpea mosaic virus is formed by 60 copies of the large (L) and small (S) coat protein subunits. The 24-amino-acid C-terminal peptide of the S coat protein can undergo proteolytic cleavage without affecting particle stability or infectivity. Mutagenic studies have shown that this sequence is involved in particle assembly, virus movement, RNA encapsidation and suppression of gene silencing. However, it is unclear how these processes are related, and which part(s) of the sequence are involved in each process. Here, we have analysed the effect of mutations in the C-terminal region of the S protein on the assembly of empty virus-like particles and on the systemic movement of infectious virus. The results confirmed the importance of positively charged amino acids adjacent to the cleavage site for particle assembly and revealed that the C-terminal 11 amino acids are important for efficient systemic movement of the virus.

Revealing the density of encoded functions in a viral RNA.

We present direct experimental evidence that assembly of a single-stranded RNA virus occurs via a packaging signal-mediated mechanism. We show that the sequences of coat protein recognition motifs within multiple, dispersed, putative RNA packaging signals, as well as their relative spacing within a genomic fragment, act collectively to influence the fidelity and yield of capsid self-assembly in vitro. These experiments confirm that the selective advantages for viral yield and encapsidation specificity, predicted from previous modeling of packaging signal-mediated assembly, are found in Nature. Regions of the genome that act as packaging signals also function in translational and transcriptional enhancement, as well as directly coding for the coat protein, highlighting the density of encoded functions within the viral RNA. Assembly and gene expression are therefore direct molecular competitors for different functional folds of the same RNA sequence. The strongest packaging signal in the test fragment, encodes a region of the coat protein that undergoes a conformational change upon contact with packaging signals. A similar phenomenon occurs in other RNA viruses for which packaging signals are known. These contacts hint at an even deeper density of encoded functions in viral RNA, which if confirmed, would have profound consequences for the evolution of this class of pathogens.

Combining high-resolution cryo-electron microscopy and mutagenesis to develop cowpea mosaic virus for bionanotechnology.

Particles of cowpea mosaic virus (CPMV) have enjoyed considerable success as nanoparticles. The development of a system for producing empty virus-like particles (eVLPs) of the virus, which are non-infectious and have the potential to be loaded with heterologous material, has increased the number of possible applications for CPMV-based particles. However, for this potential to be realised, it was essential to demonstrate that eVLPs were accurate surrogates for natural virus particles, and this information was provided by high-resolution cryo-EM studies of eVLPs. This demonstration has enabled the approaches developed for the production of modified particles developed with natural CPMV particles to be applied to eVLPs. Furthermore, a combination of cryo-EM and mutagenic studies allowed the development of particles which are permeable but which could still assemble efficiently. These particles were shown to be loadable with cobalt, indicating that they can, indeed, be used as nano-containers.

Modularity of plant metabolic gene clusters: a trio of linked genes that are collectively required for acylation of triterpenes in oat.

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis.

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants.

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, ß-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate ß-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13ß-epoxy-3ß,16ß-dihydroxy-oleanane (12,13ß-epoxy-16ß-hydroxy-ß-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family--as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism.

When plant virology met Agrobacterium: the rise of the deconstructed clones.

In the early days of molecular farming, Agrobacterium-mediated stable genetic transformation and the use of plant virus-based vectors were considered separate and competing technologies with complementary strengths and weaknesses. The demonstration that 'agroinfection' was the most efficient way of delivering virus-based vectors to their target plants blurred the distinction between the two technologies and permitted the development of 'deconstructed' vectors based on a number of plant viruses. The tobamoviruses, potexviruses, tobraviruses, geminiviruses and comoviruses have all been shown to be particularly well suited to the development of such vectors in dicotyledonous plants, while the development of equivalent vectors for use in monocotyledonous plants has lagged behind. Deconstructed viral vectors have proved extremely effective at the rapid, high-level production of a number of pharmaceutical proteins, some of which are currently undergoing clinical evaluation.

Applications of plant viruses in bionanotechnology.

The capsids of most plant viruses are simple and robust structures consisting of multiple copies of one or a few types of protein subunit arranged with either icosahedral or helical symmetry. In many cases, capsids can be produced in large quantities either by the infection of plants or by the expression of the subunit(s) in a variety of heterologous systems. In view of their relative simplicity, stability and ease of production, plant virus particles or virus-like particles (VLPs) have attracted attention as potential reagents for applications in bionanotechnology. As a result, plant virus particles have been subjected to both genetic and chemical modification, have been used to encapsulate foreign material and have, themselves, been incorporated into supramolecular structures.

Fine-tuning levels of heterologous gene expression in plants by orthogonal variation of the untranslated regions of a nonreplicating transient expression system.

A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA-2, termed CPMV-HT, in which the sequence to be expressed is positioned between a modified 5' UTR and the 3' UTR has been successfully used for the plant-based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5' UTR can dramatically influence expression levels, the role of the 3' UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3'UTR of CPMV RNA-2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress-HT-GFP. The results showed that the presence of a 3' UTR in the CPMV-HT system is important for achieving maximal expression levels. Removal of the entire 3' UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y-shaped secondary structure formed by nucleotides 125-165 of the 3' UTR plays a key role in its function; mutations that disrupt this Y-shaped structure have an effect equivalent to the deletion of the entire 3' UTR. Our results suggest that the Y-shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5' and 3' UTRs in CPMV-HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.

The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants.

The pEAQ vectors are a series of plasmids designed to allow easy and quick production of recombinant proteins in plants. Their main feature is the use of the Cowpea Mosaic Virus hypertranslational "CPMV-HT" expression system, which provides high yields of recombinant protein through extremely high translational efficiency without the need for viral replication. Since their creation, the pEAQ vectors have been used to produce a wide variety of proteins in plants. Viral proteins and Virus-Like Particles (VLPs) have been of particular interest, but other types of proteins including active enzymes have also been expressed. While the pEAQ vectors have mostly been used in a transient expression context, through agroinfiltration of leaves, they have also been shown to be suitable for the production of stably transformed lines of both cell cultures and whole plants. This paper looks back on the genesis of the pEAQ vectors and reviews their use so far.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.