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In 2022, the global outbreak of monkeypox virus (MPXV) with increased human-to-human transmission triggered urgent public health interventions. Plant-derived monoclonal antibodies (mAbs) are being explored as potential therapeutic strategies due to their diverse mechanisms of antiviral activity. MPXV produces two key infectious particles: the mature virion (MV) and the extracellular enveloped virion (EV), both essential for infection and spread. Effective therapies must target both to halt replication and transmission. Our prior research demonstrated the development of a potent neutralizing mAb against MPXV MV. This study focuses on developing a plant-derived mAb targeting MPXV EV, which is critical for viral dissemination within the host and generally resistant to antibody neutralization. Our findings reveal that the mAb (H2) can be robustly produced in Nicotiana benthamiana plants via transient expression. The plant-made H2 mAb effectively targets MPXV EV by binding specifically to the A35 MPXV antigen. Importantly, H2 mAb shows notable neutralizing activity against the infectious MPXV EV particle. This investigation is the first to report the development of a plant-derived anti-EV mAb for MPXV prevention and treatment, as well as the first demonstration of anti-MPXV EV activity by an mAb across any production platform. It highlights the potential of plant-produced mAbs as therapeutics for emerging infectious diseases, including the MPXV outbreak.

Research on the use of plants for the production of vaccine antigens has increased greatly since the seminal studies published in the early 1990s. The initial strategy focused on oral delivery of minimally processed edible material from stably transgenic plants. Although substantial regulatory concerns exist regarding the use of food crops for vaccine production, the potential for vaccines expressed in seeds of corn and rice is especially promising. However, more recent development of robust transient expression systems greatly improved the potential to use plants for expression purification of recombinant antigens for more typical injectable delivery. Significant industrial development of methods for production of plant‐made vaccines rapidly and at large‐scale demonstrates the exciting capabilities of the technology. Notably, transient expression in leaves of Nicotiana benthamiana shows great potential for the production of several vaccines for human viral pathogens. Future work will address issues of processing plant material for optimal oral or parenteral delivery.

Over the last three decades, the expression of recombinant proteins in plants and plant cells has been promoted as an alternative cost-effective production platform. However, the market is still dominated by prokaryotic and mammalian expression systems, the former offering high production capacity at a low cost, and the latter favored for the production of complex biopharmaceutical products. Although plant systems are now gaining widespread acceptance as a platform for the larger-scale production of recombinant proteins, there is still resistance to commercial uptake. This partly reflects the relatively low yields achieved in plants, as well as inconsistent product quality and difficulties with larger-scale downstream processing. Furthermore, there are only a few cases in which plants have demonstrated economic advantages compared to established and approved commercial processes, so industry is reluctant to switch to plant-based production. Nevertheless, some plant-derived proteins for research or cosmetic/pharmaceutical applications have reached the market, showing that plants can excel as a competitive production platform in some niche areas. Here, we discuss the strengths of plant expression systems for specific applications, but mainly address the bottlenecks that must be overcome before plants can compete with conventional systems, enabling the future commercial utilization of plants for the production of valuable proteins.

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N -glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N -glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N -glycan sialylation. Since sialic acids are a common terminal modification on human N- glycans, the property to perform N -glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N -glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N -glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N- glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

Plants provide not only food and feed, but also herbal medicines and various raw materials for industry. Moreover, plants can be green factories producing high value bioproducts such as biopharmaceuticals and vaccines. Advantages of plant-based production platforms include easy scale-up, cost effectiveness, and high safety as plants are not hosts for human and animal pathogens. Plant cells perform many post-translational modifications that are present in humans and animals and can be essential for biological activity of produced recombinant proteins. Stimulated by progress in plant transformation technologies, substantial efforts have been made in both the public and the private sectors to develop plant-based vaccine production platforms. Recent promising examples include plant-made vaccines against COVID-19 and Ebola. The COVIFENZ® COVID-19 vaccine produced in Nicotiana benthamiana has been approved in Canada, and several plant-made influenza vaccines have undergone clinical trials. In this review, we discuss the status of vaccine production in plants and the state of the art in downstream processing according to good manufacturing practice (GMP). We discuss different production approaches, including stable transgenic plants and transient expression technologies, and review selected applications in the area of human and veterinary vaccines. We also highlight specific challenges associated with viral vaccine production for different target organisms, including lower vertebrates (e.g., farmed fish), and discuss future perspectives for the field.

Plant-made biotherapeutics are gathering momentum and some plant glycoproteins are allergens. Glycans with core β1-2xylose and α1,3fucose motifs and antennae terminated by mannose residues (e.g.: MMXF) are found on several plant allergens and can cross-react with glyco-epitopes from other sources. To date, reactivity to these cross-reactive determinants has not been associated with clinical symptoms. We produced VLP vaccines bearing the hemagglutinin(HA) of H5(A/Indonesia/5/05) or H1(A/California/07/09) influenza viruses by transfection of Nicotiana benthamiana. Subjects enrolled in Phase I/II trials were followed for evidence of allergy/hypersensitivity and development of antibodies against plant glyco-epitopes. A total of 280/349 subjects received either one (H1) or 2 doses (H5) of vaccine (5–45μg of HA/dose) intramuscularly including 40 with pre-existing plant allergies. Subjects were monitored for 6 months. IgG and IgE to plant glyco-epitopes were measured by ELISA using corn-/egg-derived avidin and bromelain as target antigens. No subject developed allergic/hypersensitivity symptoms. Some (34%) developed transient IgG and, in some cases IgE, to plant glyco-epitopes but no subject mounted an IgE response to the MMXF motif. Antibodies returned to baseline by 6 months in most subjects. VLP vaccines bearing influenza HA glycoproteins can elicit transient IgG and, in some cases, IgE responses that are not associated with either the development or worsening of allergic/hypersensitivity symptoms.

Glycoproteins are the dominant category among approved biopharmaceuticals, indicating their importance as therapeutic proteins. Glycoproteins are decorated with carbohydrate structures (or glycans) in a process called glycosylation. Glycosylation is a post-translational modification that is present in all kingdoms of life, albeit with differences in core modifications, terminal glycan structures, and incorporation of different sugar residues. Glycans play pivotal roles in many biological processes and can impact the efficacy of therapeutic glycoproteins. The majority of biopharmaceuticals are based on human glycoproteins, but non-human glycoproteins, originating from for instance parasitic worms (helminths), form an untapped pool of potential therapeutics for immune-related diseases and vaccine candidates. The production of sufficient quantities of correctly glycosylated putative therapeutic helminth proteins is often challenging and requires extensive engineering of the glycosylation pathway. Therefore, a flexible glycoprotein production system is required that allows straightforward introduction of heterologous glycosylation machinery composed of glycosyltransferases and glycosidases to obtain desired glycan structures. The glycome of plants creates an ideal starting point for N - and O -glyco-engineering of helminth glycans. Plants are also tolerant toward the introduction of heterologous glycosylation enzymes as well as the obtained glycans. Thus, a potent production platform emerges that enables the production of recombinant helminth proteins with unusual glycans. In this review, we discuss recent advances in plant glyco-engineering of potentially therapeutic helminth glycoproteins, challenges and their future prospects.

Molecular pharming is a cost-effective platform for the production of recombinant proteins in plants. Although the biopharmaceutical industry still relies on a small number of standardized fermentation-based technologies for the production of recombinant proteins there is now a greater awareness of the advantages of molecular pharming particularly in niche markets. Here we discuss some of the technical, economic and regulatory barriers that constrain the clinical development and commercialization of plant-derived pharmaceutical proteins. We also discuss strategies to increase productivity and product quality/homogeneity. The advantages of whole plants should be welcomed by the industry because this will help to reduce the cost of goods and therefore expand the biopharmaceutical market into untapped sectors.

The transmission of HIV can be prevented by the application of neutralizing monoclonal antibodies and lectins. Traditional recombinant protein manufacturing platforms lack sufficient capacity and are too expensive for developing countries, which suffer the greatest disease burden. Plants offer an inexpensive and scalable alternative manufacturing platform that can produce multiple components in a single plant, which is important because multiple components are required to avoid the rapid emergence of HIV-1 strains resistant to single microbicides. Furthermore, crude extracts can be used directly for prophylaxis to avoid the massive costs of downstream processing and purification. We investigated whether rice could simultaneously produce three functional HIV-neutralizing proteins (the monoclonal antibody 2G12, and the lectins griffithsin and cyanovirin-N). Preliminary in vitro tests showed that the cocktail of three proteins bound to gp120 and achieved HIV-1 neutralization. Remarkably, when we mixed the components with crude extracts of wild-type rice endosperm, we observed enhanced binding to gp120 in vitro and synergistic neutralization when all three components were present. Extracts of transgenic plants expressing all three proteins also showed enhanced in vitro binding to gp120 and synergistic HIV-1 neutralization. Fractionation of the rice extracts suggested that the enhanced gp120 binding was dependent on rice proteins, primarily the globulin fraction. Therefore, the production of HIV-1 microbicides in rice may not only reduce costs compared to traditional platforms but may also provide functional benefits in terms of microbicidal potency.

Enterohemorrhagic Escherichia coli -contaminated food products are among the leading causes of bacterial enteric infections in the United States and worldwide. Currently, other than thermal inactivation, there are no effective methods to control pathogenic bacteria in food. We investigated colicins, nonantibiotic antimicrobial proteins produced by certain E. coli strains and active against other strains of the species, as potential pathogen control agents. We demonstrate that most colicins can be expressed at high yields in plants and are fully functional. We show that mixtures of colicins applied at low concentrations are highly and broadly active against all major pathogenic E. coli strains of concern for foodborne illness. We propose plant-produced colicins as an inexpensive food treatment for the broad control of pathogenic E. coli strains. Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.