In December 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in Wuhan, China, leading to the global Coronavirus Disease pandemic. The rapid spread of SARS-CoV-2 highlighted the urgent need for effective vaccines. However, the high cost, cold storage requirements, and scalability challenges associated with mRNA vaccines have necessitated alternative vaccine technologies. In the study, the safety of a plant-based vaccine was evaluated. The vaccine, an emulsion of the SARS-CoV-2 S1 antigen and a synthetic TLR4 agonist produced and purified from Nicotiana benthamiana, was administered to Sprague-Dawley rats three times over 4 wk. Mortality, clinical signs, body weight, food consumption, vision, urinalysis, gross findings, organ weight, hematology, serum biochemistry, histopathology, and immunogenicity were evaluated. The results showed that antibodies were efficiently produced and maintained for one month following vaccination with the plant-derived receptor-binding domain (RBD) antigen of COVID-19. Furthermore, the rats showed no toxicological symptoms, with reversible changes at the injection site and minor histological alterations in the spinal cord and bone marrow, typical of vaccine responses. The plant-derived SARS-CoV-2 vaccine appears safe following repeated administration over 4 wk and represents a promising alternative for potential use in human clinical trials and clinical applications.

The plant-based system is a promising platform for producing biotherapeutics due to its scalability, cost-effectiveness, and lower risk of contamination by human pathogens. However, several challenges remain, including optimizing yield, stability, functionality, and the immunogenic properties of recombinant proteins. In this context, this review explores the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology to improve the production of recombinant therapeutic proteins in plants. Traditional tools and strategies for plant-based recombinant protein production are discussed, highlighting their limitations and the potential of CRISPR to overcome these boundaries. It delves into the components of the CRISPR-Cas system and its application in optimizing therapeutic protein function and yield. Major strategies include modifying glycosylation patterns to humanize plant-produced proteins, metabolic pathway engineering to increase protein accumulation, and the precise integration of transgenes into specific genomic loci to enhance expression stability and productivity. These advancements demonstrate how CRISPR system can overcome bottlenecks in plant molecular farming and enable the production of high-quality therapeutic proteins. Lastly, future trends and perspectives are examined, emphasizing ongoing innovations and challenges in the field. The review underscores the potential of CRISPR to reshape plant biotechnology and support the growing demand for recombinant therapeutics, offering new avenues for sustainable and efficient protein production systems. KEY MESSAGE: CRISPR technology has the potential to improve plant-based therapeutic protein production by optimizing yield, stability, and humanization, overcoming bottlenecks, and enabling sustainable, efficient systems for recombinant biotherapeutics.

Molecular farming, initially developed to produce therapeutic proteins using genetically modified plants, gained renewed interest during the Ebola and COVID-19 outbreaks and has expanded into functional food ingredients. This article evaluates molecular farming technologies, market potential, and startups, and identifies opportunities in dairy proteins, food enzymes, collagen, and cellular agriculture.

Antimicrobial peptides (AMPs) are critical effectors of innate immunity, presenting a compelling alternative to conventional antibiotics amidst escalating antimicrobial resistance. Their broad-spectrum efficacy and inherent low resistance development are countered by production challenges, including limited yields and proteolytic degradation, which restrict their clinical translation. While chemical synthesis offers precise structural control, it is often prohibitively expensive and complex for large-scale production. Heterologous expression systems provide a scalable, cost-effective platform, but necessitate optimization. This review comprehensively examines established and emerging AMP production strategies, encompassing fusion protein technologies, molecular engineering approaches, rational peptide design, and post-translational modifications, with an emphasis on maximizing yield, bioactivity, stability, and safety. Furthermore, we underscore the transformative role of artificial intelligence, particularly machine learning algorithms, in accelerating AMP discovery and optimization, thereby propelling their expanded therapeutic application and contributing to the global fight against drug-resistant infections.

The COVID-19 pandemic highlights the opportunity for mRNA vaccines and their nanotechnology carriers to make an impact as a countermeasure to infectious disease. As alternative to the synthetic lipid nanoparticles or mammalian viruses, we developed a tobacco mosaic virus (TMV)-based mRNA vaccine delivery platform. Specifically, purified coat protein from TMV was used to package a self-amplifying Nodamura replicon expressing the receptor binding domain (RBD) from the Omicron strain of SARS-CoV-2. The replicon construct contains the origin of assembly sequence from the tobacco mosaic virus (TMV) for encapsulation and mRNA stabilization. The nanoparticle vaccine was obtained through in vitro assembly using purified TMV coat proteins and in vitro transcribed mRNA cassettes. Cell assays confirmed delivery of self-amplifying mRNA vaccine, amplification of the transgene and expression of the target protein, RBD, in mammalian cells. Immunization of mice yielded RBD-specific IgG antibodies that demonstrated neutralization of SARS-CoV-2 using an in vitro neutralization assay. The TMV platform nanotechnology does not require ultralow freezers for storage or distribution; and the in vitro assembly method provide 'plug-and-play' to adapt the vaccine formulation rapidly as new strains or diseases emerge. Finally, opportunity exists to produce and self-assemble the vaccine candidate in plants through molecular farming techniques, which may allow production in the region-for the region and could make a contribution to less resourced areas of the world.

The production of coronavirus disease 2019 vaccines can be achieved by transient expression of the spike (S) protein of severe acute respiratory syndrome coronavirus 2 in agroinfiltrated leaves of Nicotiana benthamiana. Relying on bacterial vector Agrobacterium tumefaciens, this process is favoured by co-expression of viral silencing suppressor P19. Upon expression, the S protein enters the cell secretory pathway, before being trafficked to the plasma membrane where formation of coronavirus-like particles (CoVLPs) occurs. We previously characterized the effects of influenza virus hemagglutinin forming VLPs through similar processes. However, leaf samples were only collected after 6 days of expression, and it is unknown whether influenza VLPs (HA-VLPs) and CoVLPs induce similar responses. Here, time course sampling was used to profile responses of N. benthamiana leaf cells expressing P19 only, or P19 and the S protein. The latter triggered early but transient activation of the unfolded protein response and waves of transcription factor genes involved in immunity. Accordingly, defence genes were induced with different expression kinetics, including those promoting lignification, terpene biosynthesis, and oxidative stress. Cross-talk between stress hormone pathways also occurred, including repression of jasmonic acid biosynthesis genes after agroinfiltration, and dampening of salicylic acid responses upon S protein accumulation. Overall, HA-VLP- and CoVLP-induced responses broadly overlapped, suggesting nanoparticle production to have the most effects on plant immunity, regardless of the virus surface proteins expressed. Taking advantage of RNAseq inferences, we finally show the co-expression of Kunitz trypsin inhibitors to reduce CoVLP-induced defence and leaf symptoms, with no adverse effect on plant productivity.

Enteric diseases by pathogenic organisms are one of the leading causes of death worldwide, particularly in low-income countries. Despite antibiotics, access to clean water and vaccination are the most economically affordable options to prevent those infections and their health consequences. Vaccines, such as those approved for rotavirus and cholera, have played a key role in preventing several enteric diseases. However, vaccines for other pathogens are still in clinical trials. Distribution and cost remain significant barriers to vaccine access in developing regions due to poor healthcare infrastructure, cold-chain requirements, and high production costs. Plant-made vaccines offer a promising alternative to address these challenges. Plants can be easily grown, lowering production costs, and can be administered in oral forms, potentially eliminating cold-chain dependency. Although there are some promising prototypes of vaccines produced in plants, challenges remain, including yields and achieving sufficient immunogenicity. This review aims to describe common enteric pathogens and available vaccines, followed by a strategic summary of plant-made vaccine development and a discussion of plant-made enteric vaccine prototypes. Trends to overcome the key challenges for plant-made vaccines are identified and placed in perspective for the development of affordable and effective vaccines for populations at the highest risk of enteric diseases.

Using plants as bioreactors, molecular farming has emerged as a versatile and sustainable platform for producing recombinant vaccines, therapeutic proteins, industrial enzymes, and nutraceuticals. This innovative approach leverages the unique advantages of plants, including scalability, cost-effectiveness, and reduced risk of contamination with human pathogens. Recent advancements in gene editing, transient expression systems, and nanoparticle-based delivery technologies have significantly enhanced the efficiency and versatility of plant-based systems. Particularly in vaccine development, molecular farming has demonstrated its potential with notable successes such as Medicago's Covifenz for COVID-19, illustrating the capacity of plant-based platforms to address global health emergencies rapidly. Furthermore, edible vaccines have opened new avenues in the delivery of vaccines, mainly in settings with low resources where the cold chain used for conventional logistics is a challenge. However, optimization of protein yield and stability, the complexity of purification processes, and regulatory hurdles are some of the challenges that still remain. This review discusses the current status of vaccine development using plant-based expression systems, operational mechanisms for plant expression platforms, major applications in the prevention of infectious diseases, and new developments, such as nanoparticle-mediated delivery and cancer vaccines. The discussion will also touch on ethical considerations, the regulatory framework, and future trends with respect to the transformative capacity of plant-derived vaccines in ensuring greater global accessibility and cost-effectiveness of the vaccination. This field holds great promise for the infectious disease area and, indeed, for applications in personalized medicine and biopharmaceuticals in the near future.

Maize, a cornerstone of global food security, has undergone remarkable transformations through breeding, yet further increase in global maize production faces mounting challenges in a changing world. In this Perspective paper, we overview the historical successes of maize breeding that laid the foundation for present opportunities. We examine both the specific and shared breeding goals related to diverse geographies and end-use demands. Achieving these coordinated breeding objectives requires a holistic approach to trait improvement for sustainable agriculture. We discuss cutting-edge solutions, including multi-omics approaches from single-cell analysis to holobionts, smart breeding with advanced technologies and algorithms, and the transformative potential of rational design with synthetic biology approaches. A transition toward a data-driven future is currently underway, with large-scale precision agriculture and autonomous systems poised to revolutionize farming practice. Realizing these futuristic opportunities hinges on collaborative efforts spanning scientific discoveries, technology translations, and socioeconomic considerations in maximizing human and environmental well-being.

The growing demand for sustainable platforms for biomolecule manufacturing has fuelled the development of plant-based production systems. Agroinfiltration, the current industry standard, offers several advantages but faces limitations for large-scale production due to high operational costs and batch-to-batch variability. Alternatively, here, we describe the CuBe system, a novel bean yellow dwarf virus (BeYDV)-derived conditional replicative expression platform stably transformed in Nicotiana benthamiana and activated by copper sulphate (CuSO4), an inexpensive and widely used agricultural input. The CuBe system utilizes a synthetic circuit of four genetic modules integrated into the plant genome: (i) a replicative vector harbouring the gene of interest (GOI) flanked by cis-acting elements for geminiviral replication and novelly arranged to enable transgene transcription exclusively upon formation of the circular replicon, (ii) copper-inducible Rep/RepA proteins essential for replicon formation, (iii) the yeast-derived CUP2-Gal4 copper-responsive transcriptional activator for Rep/RepA expression, and (iv) a copper-inducible Flp recombinase to minimize basal Rep/RepA expression. CuSO4 application triggers the activation of the system, leading to the formation of extrachromosomal replicons, expression of the GOI, and accumulation of the desired recombinant protein. We demonstrate the functionality of the CuBe system in N. benthamiana plants expressing high levels of eGFP and an anti-SARS-CoV-2 antibody upon copper treatment. Notably, the system is functional in post-harvest applications, a strategy with high potential impact for large-scale biomanufacturing. This work presents the CuBe system as a promising alternative to agroinfiltration for cost-effective and scalable production of recombinant proteins in plants.

Viral nanoparticles (VNPs) have emerged as crucial tools in the field of biomedicine. Leveraging their biological and physicochemical properties, VNPs exhibit significant advantages in the prevention, diagnosis, and treatment of human diseases. Through techniques such as chemical bioconjugation, infusion, genetic engineering, and encapsulation, these VNPs have been endowed with multifunctional capabilities, including the display of functional peptides or proteins, encapsulation of therapeutic drugs or inorganic particles, integration with imaging agents, and conjugation with bioactive molecules. This review provides an in-depth analysis of VNPs in biomedicine, elucidating their diverse types, distinctive features, production methods, and complex design principles behind multifunctional VNPs. It highlights recent innovative research and various applications, covering their roles in imaging, drug delivery, therapeutics, gene delivery, vaccines, immunotherapy, and tissue regeneration. Additionally, the review provides an assessment of their safety and biocompatibility and discusses challenges and future opportunities in the field, underscoring the vast potential and evolving nature of VNP research.

Viral nanoparticles (VNPs) are self-assembled nanometric complexes whose size and shape are similar to those of the virus from which they are derived. VNPs are arousing great attention due to potential biotechnological applications in fields like nanomedicine and nanotechnology because they allow the presentation of polypeptides of choice linked to the virus structural proteins. Starting from tobacco etch virus (TEV), a plant plus-strand RNA virus that belongs to the genus Potyvirus (family Potyviridae), here we describe the development of recombinant hybrid VNPs in Nicotiana benthamiana plants able of exposing simultaneously different proteins on their surface. This system is based on the synergic coinfection of TEV and potato virus X (PVX; Potexvirus), in which PVX provides a second TEV CP in trans allowing a mixed assembly. We first generated genetically modified hybrid VNPs simultaneously displaying green and red fluorescent proteins on their surface. A population of decorated and nondecorated CPs resulting from the insertion of the picornavirus F2A ribosomal escape peptide was required for viral particle assembly. Correct assembly of the recombinant mosaic VNPs presenting the exogenous peptides was successfully observed by immunoelectron microscopy. We next achieved the production of hybrid VNPs expressing a nanobody against SARS-CoV-2 and a fluorescent reporter protein, whose functionality was demonstrated by ELISA and dot-blot assay. Finally, we engineered the production of hybrid multivalent VNPs carrying two different nanobodies against distinct epitopes of the same SARS-CoV-2 antigenic protein, emulating a nanobody cocktail. These plant-produced recombinant mosaic VNPs, which are filamentous and flexuous in shape, presenting two different fused proteins on the surface, represent a molecular tool with several potential applications in biotechnology.

Bioactive peptides and proteins (BAPPs) are promising therapeutic agents for tissue repair with considerable advantages, including multifunctionality, specificity, biocompatibility, and biodegradability. However, the high complexity of tissue microenvironments and their inherent deficiencies such as short half-live and susceptibility to enzymatic degradation, adversely affect their therapeutic efficacy and clinical applications. Investigating the fundamental mechanisms by which BAPPs modulate the microenvironment and developing rational delivery strategies are essential for optimizing their administration in distinct tissue repairs and facilitating clinical translation. This review initially focuses on the mechanisms through which BAPPs influence the microenvironment for tissue repair via reactive oxygen species, blood and lymphatic vessels, immune cells, and repair cells. Then, a variety of delivery platforms, including scaffolds and hydrogels, electrospun fibers, surface coatings, assisted particles, nanotubes, two-dimensional nanomaterials, and nanoparticles engineered cells, are summarized to incorporate BAPPs for effective tissue repair, modification strategies aimed at enhancing loading efficiencies and release kinetics are also reviewed. Additionally, the delivery of BAPPs can be precisely regulated by endogenous stimuli (glucose, reactive oxygen species, enzymes, pH) or exogenous stimuli (ultrasound, heat, light, magnetic field, and electric field) to achieve on-demand release tailored for specific tissue repair needs. Furthermore, this review focuses on the clinical potential of BAPPs in facilitating tissue repair across various types, including bone, cartilage, intervertebral discs, muscle, tendons, periodontal tissues, skin, myocardium, nervous system (encompassing brain, spinal cord, and peripheral nerve), endometrium, as well as ear and ocular tissue. Finally, current challenges and prospects are discussed.

Viruses can be designed to be tools and carrier vehicles for intratumoural immunotherapy. Their nanometre-scale size and shape allow for functionalization with or encapsulation of medical cargoes and tissue-specific ligands. Importantly, immunotherapies may particularly benefit from the inherent immunomodulatory properties of viruses. For example, mammalian viruses have already been tested for oncolytic virotherapy, and bacteriophages and plant viruses can be engineered for immunotherapeutic treatment approaches. In this Review, we discuss how viruses - including oncolytic viruses, immunomodulatory plant viruses and bacteriophages - and virus-like particles can be designed for intratumoural immunotherapy to elicit anti-tumour immunity and induce systemic anti-tumour responses at distant non-injected sites. We further highlight the engineering of viruses and virus-like particles as drug-delivery systems, and outline key translational challenges and clinical opportunities.

Plant-made pharmaceuticals (PMP) have great potential in terms of production costs, scalability, safety, environmental protection, and consumer acceptability. The first PMP were antibodies and antigens produced in stably transformed transgenic plants in the around 90s. Even though the effort using stable transgenic plants is still going on, the mainstream of PMP production has shifted to transient expression in Nicotiana benthamiana. This system involves the expression vectors by Agrobacterium, and its efficiency has been improved by the development of new vector systems and host engineering. The COVID-19 outbreak accelerated this trend through efforts to produce vaccines in plants. Transient expression systems have been improved and diversified by the development of plant virus vectors, which can be classified as full and deconstructed vectors. Full virus vectors spread systemically, allowing for protein production in the entire plant. Compared with conventional agroinfiltration vectors, excellent virus vectors result in higher protein production. Engineering of host plants has included knocking out gene-silencing systems to increase protein production, and the introduction of glycan modification enzymes so that plant-made proteins more resemble animal-made proteins. Hydroponic cultivation systems in plant factories and environmental controls have contributed to efficient protein production in plants. Considering their advantages and small environmental impact, PMP should be more widely adopted for pharmaceuticals' production. However, the initial investment and running costs of plant factories are higher than open filed cultivation. The next objectives are to develop next-generation low-cost plant factories that use renewable energy and recycle materials based on the idea of circular economy.

In this work, we introduce a 3D-printable virus-like particle (VLP)-enhanced cross-linked biopolymer system. VLPs displaying surface-available acrylate groups were prepared through aza-Michael addition to serve as resins. The VLP resins were then photopolymerized into a poly(ethylene glycol) diacrylate (PEGDA) network following DLP 3D printing. This approach represents a convergence of disciplines, where the synergistic interaction between virology and additive manufacturing unlocks new frontiers in biotechnology.

Antimicrobial peptides (AMPs) are emerging as next-generation therapeutics due to their broad-spectrum activity against drug-resistant bacterial strains and their ability to eradicate biofilms, modulate immune responses, exert anti-inflammatory effects and improve disease management. They are produced through solid-phase peptide synthesis or in bacterial or yeast cells. Molecular farming, i.e. the production of biologics in plants, offers a low-cost, non-toxic, scalable and simple alternative platform to produce AMPs at a sustainable cost. In this review, we discuss the advantages of molecular farming for producing clinical-grade AMPs, advances in expression and purification systems and the cost advantage for industrial-scale production. We further review how 'green' production is filling the sustainability gap, streamlining patent and regulatory approvals and enabling successful clinical translations that demonstrate the future potential of AMPs produced by molecular farming. Finally, we discuss the regulatory challenges that need to be addressed to fully realize the potential of molecular farming-based AMP production for therapeutics.

Protein cages are promising tools for the controlled delivery of therapeutics and imaging agents when endowed with programmable disassembly strategies. Here, we produced hybrid nanocomposites made of tobacco mosaic virus (TMV) and magnetic iron oxide nanoparticles (IONPs), designed to disrupt the viral protein cages using magnetically induced release of heat. We studied the effects of this magnetic hyperthermia on the programmable viral protein capsid disassembly using (1) elongated nanocomposites of TMV coated heterogeneously with magnetic iron oxide nanoparticles (TMV@IONPs) and (2) spherical nanocomposites of polystyrene (PS) on which we deposited presynthesized IONPs and TMV via layer-by-layer self-assembly (PS@IONPs/TMV). Notably, we found that the extent of the disassembly of the protein cages is contingent upon the specific absorption rate (SAR) of the magnetic nanoparticles, that is, the heating efficiency, and the relative position of the protein cage within the nanocomposite concerning the heating sources. This implies that the spatial arrangement of components within the hybrid nanostructure has a significant impact on the disassembly process. Understanding and optimizing this relationship will contribute to the critical spatiotemporal control for targeted drug and gene delivery using protein cages.

Excessive exercise is an etiological factor of intervertebral disc degeneration (IVDD). Engineered extracellular vesicles (EVs) exhibit excellent therapeutic potential for disease-modifying treatments. Herein, we fabricate an exercise self-powered triboelectric-responsive microneedle (MN) assay with the sustainable release of optogenetically engineered EVs for IVDD repair. Mechanically, exercise promotes cytosolic DNA sensing-mediated inflammatory activation in senescent nucleus pulposus (NP) cells (the master cell population for IVD homeostasis maintenance), which accelerates IVDD. TREX1 serves as a crucial nuclease, and disassembly of TRAM1-TREX1 complex disrupts the subcellular localization of TREX1, triggering TREX1-dependent genomic DNA damage during NP cell senescence. Optogenetically engineered EVs deliver TRAM1 protein into senescent NP cells, which effectively reconstructs the elimination function of TREX1. Triboelectric nanogenerator (TENG) harvests mechanical energy and triggers the controllable release of engineered EVs. Notably, an optogenetically engineered EV-based targeting treatment strategy is used for the treatment of IVDD, showing promising clinical potential for the treatment of degeneration-associated disorders.

Tobacco mild green mosaic virus (TMGMV)-like nanocarriers were designed for gene delivery to plant cells. High aspect ratio TMGMVs were coated with a polycationic biopolymer, poly(allylamine) hydrochloride (PAH), to generate highly charged nanomaterials (TMGMV-PAH; 56.20 ± 4.7 mV) that efficiently load (1:6 TMGMV:DNA mass ratio) and deliver single-stranded and plasmid DNA to plant cells. The TMGMV-PAH were taken up through energy-independent mechanisms in Arabidopsis protoplasts. TMGMV-PAH delivered a plasmid DNA encoding a green fluorescent protein (GFP) to the protoplast nucleus (70% viability), as evidenced by GFP expression using confocal microscopy and Western blot analysis. TMGMV-PAH were inactivated (iTMGMV-PAH) using UV cross-linking to prevent systemic infection in intact plants. Inactivated iTMGMV-PAH-mediated pDNA delivery and gene expression of GFP in vivo was determined using confocal microscopy and RT-qPCR. Virus-like nanocarrier-mediated gene delivery can act as a facile and biocompatible tool for advancing genetic engineering in plants.

Hypothesis Long-acting formulations such as microparticles, injectable depots and implantable devices can realize spatiotemporally controlled delivery of protein drugs to extend their therapeutic in vivo half-lives. To efficiently encapsulate the protein drugs into such drug delivery systems, (sub)micron-sized protein particles are needed. The formation of micronized supraproteins can be induced through the synergistic combination of attractive depletion forces and freezing. The size of the supraproteins can be fine-tuned from submicron to several microns by adjusting the ice crystallization rate through the freeze-quench depth, which is set by the target temperature. Methods Supraprotein micron structures were prepared from protein solutions under various conditions in the presence and absence of nonadsorbing polyethylene glycol. Scanning electron microscopy and dynamic light scattering were employed to determine the sizes of the supraproteins and real-time total internal reflection fluorescent microscopy was used to follow the supraprotein formation during freezing. The protein secondary structure was measured before and after micronization by circular dichroism. A phase diagram of a protein-polyethylene glycol mixture was theoretically predicted to investigate whether the depletion interaction can elucidate the phase behavior. Findings Micronized protein supraparticles could be prepared in a controlled manner by rapid freeze-drying of aqueous mixtures of bovine serum albumin, horseradish peroxidase and lysozyme mixed with polyethylene glycol. Upon freezing, the temperature quench initiates a phase separation process which is reminiscent of spinodal decomposition. This demixing is subsequently arrested during droplet phase separation to form protein-rich microstructures. The final size of the generated protein microparticles is determined by a competition between phase separation and cooling rate, which can be controlled by target temperature. The experimental phase diagram of the aqueous protein-polyethylene glycol dispersion aligns with predictions from depletion theory for charged colloids and nonadsorbing polymers.

Bulk commodity row crop production in the United States is frequently subject to narrow profit margins, often complicated by weather, supply chains, trade, and other factors. Farmers seeking to increase profits and hedge against market volatility often seek to diversify their operations, including producing more lucrative or productive crop varieties. Recombinant plants producing animal or other non-native proteins (commonly referred to as plant molecular farming) present a value-added opportunity for row crop farmers. However, these crops must be produced under robust identity preserved systems to prevent comingling with bulk commodities to maintain the value for farmers, mitigate against market disruptions, and minimize any potential food, feed, or environmental risks.

Canine atopic dermatitis (CAD) is an allergic, inflammatory, and pruritic skin disease associated with the production of IgE antibodies against environmental allergens and mainly house dust mite allergens. This complex dermatological pathology involves Interleukin 31 (IL-31) as a central itch mediator. One of the most effective CAD treatments is a caninized monoclonal antibody (mAb) called Lokivetmab. It is produced in CHO cells and targets specifically canine IL-31 (cIL-31) and blocks its cellular messaging. This treatment has undoubtedly contributed to a breakthrough in dermatitis-related pruritus. However, its production in mammalian cells requires time-consuming procedures, high production costs, and investment. Plants are considered an emerging protein production platform for recombinant biopharmaceuticals due to their cost-effectiveness and rapidity for production. Here, we use transient expression in Nicotiana benthamiana plants to produce recombinant canine Interleukin 31 (cIL-31) and an anti-IL-31 monoclonal antibody (M1). First, we describe the production and characterization of M1 and then its activity on an IL-31-induced pruritic model in dogs compared to its commercial homolog. Dogs treated with the plant-made M1 mAb have shown similar improvements to Lokivetmab-treated ones after different challenges using canine IL-31. Furthermore, M1 injections were not associated with any side effects. These results demonstrate the safety and efficacy of this plant-made Lokivetmab biosimilar to control dogs' pruritus in a well-established model. Finally, this study shows that the plant-production platform can be utilized to produce rapidly functional mAbs and bring hope to the immunotherapy field of veterinary medicine.

The dramatic effectiveness of recent mRNA (mRNA)-based COVID vaccines delivered in lipid nanoparticles has highlighted the promise of mRNA therapeutics in general. In this report, we extend our earlier work on self-amplifying mRNAs delivered in spherical in vitro reconstituted virus-like particles (VLPs), and on drug delivery using cylindrical virus particles. In particular, we carry out separate in vitro assemblies of a self-amplifying mRNA gene in two different virus-like particles: one spherical, formed with the capsid protein of cowpea chlorotic mottle virus (CCMV), and the other cylindrical, formed from the capsid protein of tobacco mosaic virus (TMV). The mRNA gene is rendered self-amplifying by genetically fusing it to the RNA-dependent RNA polymerase (RdRp) of Nodamura virus, and the relative efficacies of cell uptake and downstream protein expression resulting from their CCMV- and TMV-packaged forms are compared directly. This comparison is carried out by their transfections into cells in culture: expressions of two self-amplifying genes, enhanced yellow fluorescent protein (EYFP) and Renilla luciferase (Luc), packaged alternately in CCMV and TMV VLPs, are quantified by fluorescence and chemiluminescence levels, respectively, and relative numbers of the delivered mRNAs are measured by quantitative real-time PCR. The cellular uptake of both forms of these VLPs is further confirmed by confocal microscopy of transfected cells. Finally, VLP-mediated delivery of the self-amplifying-mRNA in mice following footpad injection is shown by in vivo fluorescence imaging to result in robust expression of EYFP in the draining lymph nodes, suggesting the potential of these plant virus-like particles as a promising mRNA gene and vaccine delivery modality. These results establish that both CCMV and TMV VLPs can deliver their in vitro packaged mRNA genes to immune cells and that their self-amplifying forms significantly enhance in situ expression. Choice of one VLP (CCMV or TMV) over the other will depend on which geometry of nucleocapsid is self-assembled more efficiently for a given length and sequence of RNA, and suggests that these plant VLP gene delivery systems will prove useful in a wide variety of medical applications, both preventive and therapeutic.

Alpha-amylases are crucial hydrolase enzymes which have been widely used in food, feed, fermentation, and pharmaceutical industries. Methods for low-cost production of α-amylases are highly desirable. Soybean seed, functioning as a bioreactor, offers an excellent platform for the mass production of recombinant proteins for its ability to synthesize substantial quantities of proteins. In this study, we generated and characterized transgenic soybeans expressing the α-amylase AmyS from Bacillus stearothermophilus. The α-amylase expression cassettes were constructed for seed specific expression by utilizing the promoters of three different soybean storage peptides and transformed into soybean via Agrobacterium-mediated transformation. The event with the highest amylase activity reached 601 U/mg of seed flour (one unit is defined as the amount of enzyme that generates 1 micromole reducing ends per min from starch at 65 °C in pH 5.5 sodium acetate buffer). The optimum pH, optimum temperature, and the enzymatic kinetics of the soybean expressed enzyme are similar to that of the E. coli expressed enzyme. However, the soybean expressed α-amylase is glycosylated, exhibiting enhanced thermostability and storage stability. Soybean AmyS retains over 80% activity after 100 min at 75 °C, and the transgenic seeds exhibit no significant activity loss after one year of storage at room temperature. The accumulated AmyS in the transgenic seeds represents approximately 15% of the total seed protein, or about 4% of the dry seed weight. The specific activity of the transgenic soybean seed flour is comparable to many commercial α-amylase enzyme products in current markets, suggesting that the soybean flour may be directly used for various applications without the need for extraction and purification.

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed. Adaptations for distinct particle species, pitfalls, and 'forgotten' or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods.

Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected Brucella melitensis T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in Nicotiana benthamiana plants. We successfully demonstrated the assembly of five chimeric CLPs in N. benthamiana plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent Brucella strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting B. melitensis T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis.

The unfolded protein response (UPR) allows cells to cope with endoplasmic reticulum (ER) stress induced by accumulation of misfolded proteins in the ER. Due to its sensitivity to Agrobacterium tumefaciens, the model plant Nicotiana benthamiana is widely employed for transient expression of recombinant proteins of biopharmaceutical interest, including antibodies and virus surface proteins used for vaccine production. As such, study of the plant UPR is of practical significance, since enforced expression of complex secreted proteins often results in ER stress. After 6 days of expression, we recently reported that influenza haemagglutinin H5 induces accumulation of UPR proteins. Since up-regulation of corresponding UPR genes was not detected at this time, accumulation of UPR proteins was hypothesized to be independent of transcriptional induction, or associated with early but transient UPR gene up-regulation. Using time course sampling, we here show that H5 expression does result in early and transient activation of the UPR, as inferred from unconventional splicing of NbbZIP60 transcripts and induction of UPR genes with varied functions. Transient nature of H5-induced UPR suggests that this response was sufficient to cope with ER stress provoked by expression of the secreted protein, as opposed to an antibody that triggered stronger and more sustained UPR activation. As up-regulation of defence genes responding to H5 expression was detected after the peak of UPR activation and correlated with high increase in H5 protein accumulation, we hypothesize that these immune responses, rather than the UPR, were responsible for onset of the necrotic symptoms on H5-expressing leaves.

The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus-like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co-expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co-expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium-mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co-expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin-related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs.

Viral nanoparticles (VNPs) are a new class of virus-based formulations that can be used as building blocks to implement a variety of functions of potential interest in biotechnology and nanomedicine. Viral coat proteins (CP) that exhibit self-assembly properties are particularly appropriate for displaying antigens and antibodies, by generating multivalent VNPs with therapeutic and diagnostic potential. Here, we developed genetically encoded multivalent VNPs derived from two filamentous plant viruses, potato virus X (PVX) and tobacco etch virus (TEV), which were efficiently and inexpensively produced in the biofactory Nicotiana benthamiana plant. PVX and TEV-derived VNPs were decorated with two different nanobodies recognizing two different regions of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The addition of different picornavirus 2A ribosomal skipping peptides between the nanobody and the CP allowed for modulating the degree of VNP decoration. Nanobody-decorated VNPs purified from N. benthamiana tissues successfully recognized the RBD antigen in enzyme-linked immunosorbent assays and showed efficient neutralization activity against pseudoviruses carrying the Spike protein. Interestingly, multivalent PVX and TEV-derived VNPs exhibited a neutralizing activity approximately one order of magnitude higher than the corresponding nanobody in a dimeric format. These properties, combined with the ability to produce VNP cocktails in the same N. benthamiana plant based on synergistic infection of the parent PVX and TEV, make these green nanomaterials an attractive alternative to standard antibodies for multiple applications in diagnosis and therapeutics.

Many virus-like particles (VLPs) have good chemical, thermal, and mechanical stabilities compared to those of other biologics. However, their stability needs to be improved for the commercialization and use in translation of VLP-based materials. We developed an endoskeleton-armored strategy for enhancing VLP stability. Specifically, the VLPs of physalis mottle virus (PhMV) and Qβ were used to demonstrate this concept. We built an internal polymer "backbone" using a maleimide-PEG15-maleimide cross-linker to covalently interlink viral coat proteins inside the capsid cavity, while the native VLPs are held together by only noncovalent bonding between subunits. Endoskeleton-armored VLPs exhibited significantly improved thermal stability (95 °C for 15 min), increased resistance to denaturants (i.e., surfactants, pHs, chemical denaturants, and organic solvents), and enhanced mechanical performance. Single-molecule force spectroscopy demonstrated a 6-fold increase in rupture distance and a 1.9-fold increase in rupture force of endoskeleton-armored PhMV. Overall, this endoskeleton-armored strategy provides more opportunities for the development and applications of materials.

Helical morphologies are widely observed in nature, however, it is very challenging to prepare artificial helical polymers. Especially, precisely understanding the structure information of artificial metal-free helical covalent inorganic polymers via single-crystal X-ray diffraction (SCXRD) analysis is rarely explored. Here, we successfully prepare a novel metal-free helical covalent inorganic polymer ({[Te(C6 H5 )2 ] [PO3 (OH)]}n , named CityU-10) by introducing angular anions (HOPO3 2- ) into traditional tellurium-oxygen chains. The dynamic reversibility of the reaction is realized through the introduction of organic tellurium precursor and the slow hydrolysis of polyphosphoric acid. High-quality and large-size single crystals of CityU-10 have been successfully characterized via SCXRD, where the same-handed helical inorganic polymer chains form a pseudo-two-dimensional layer via multiple hydrogen-bonding interactions. The left-handed layers and right-handed layers alternatively stack together through weak hydrogen bonds to form a three-dimensional supramolecular structure. The single crystals of CityU-10 are found to display promising optical properties with a large birefringence. Our results would offer new guidelines for designing and preparing new crystalline covalent polymers through tellurium-based chemistry.

As a safe and natural "capsule," plants have several advantages over mammals and microorganisms for the production of oral vaccines. In this study, we innovatively utilized the transmembrane region of the pea Translocase of chloroplast 34 (TOC34) protein to display two subunit vaccines, capsid protein VP2 of Porcine parvovirus (PPV) and the heat-labile enterotoxin B (LTB) of Escherichia coli, on the surface of chloroplasts. Unlike microbial display techniques, chloroplast display circumvents antigen degradation in the stomach while retaining the size characteristic of microorganisms. Additionally, a co-expressed peptide adjuvant, antimicrobial peptide protegin-1 (PG1), was used to enhance the strength of oral immunization. Immunohistochemistry and trypsin digestion of chloroplast surface proteins confirmed the successful localization of both antigens on the chloroplast surface. In stable transgenic tobacco plants, the expression level of VP2-TOC34 ranged from 0.21 to 6.83 μg/g FW, while LTB-TOC34 ranged from 2.42 to 10.04 μg/g FW. By contrasting the digestive characteristics of plant materials with different particle sizes, it was observed that plant materials with diameters around 1 mm exhibited more prominent advantages in terms of chloroplast release and antigen exposure compared to both larger and smaller particles. Oral immunization resulted in significantly increased levels of specific IgG and secretory IgA in the mice compared to the control, with similar effects observed between the groups receiving oral immunization alone and those receiving a combination of initial injection and subsequent oral immunization. Challenge experiments further demonstrated the effective protection against infection in mice using this approach. These findings highlight the potential of chloroplast display technology for the development of effective oral vaccines.

Intervertebral disc degeneration (IVDD) is a global public health issue. The injury of annulus fibrosus (AF) caused by acupuncture or discectomy can trigger IVDD again. However, there is currently no suitable method for treating AF injury. In this study, the high-strength smart microneedles (MNs) which can penetrate the AF tissue through a local and minimally invasive method, and achieve remote control of speeded-up release of the drug and hyperthermia by the Near Infrared is developed. The PDA/GelMA composite MNs loaded with diclofenac sodium are designed to extracellularly "offend" the inflammatory microenvironment and mitigate damage to cells, and intracellularly increase the level of cytoprotective heat shock proteins to enhance the defense against the hostile microenvironment, achieving "offensive and defensive" effects. In vitro experiments demonstrate that the synergistic treatment of photothermal therapy and anti-inflammation effectively reduces inflammation, inhibits cell apoptosis, and promotes the synthesis of the extracellular matrix (ECM). In vivo experiments show that the MNs mitigate the inflammatory response, promote ECM deposition, reduce the level of apoptosis, and restore the biomechanical properties of the intervertebral disc (IVD) in rats. Overall, this high-strength smart MNs display promising "offensive and defensive" effects that can provide a new strategy for IVD repair.

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.

Metastatic cancer accounts for 90% of all cancer-related deaths and continues to be one of the toughest challenges in cancer treatment. A growing body of data indicates that S100A9, a major regulator of inflammation, plays a central role in cancer progression and metastasis, particularly in the lungs, where S100A9 forms a premetastatic niche. Thus, we developed a vaccine against S100A9 derived from plant viruses and virus-like particles. Using multiple tumor mouse models, we demonstrate the effectiveness of the S100A9 vaccine candidates in preventing tumor seeding within the lungs and outgrowth of metastatic disease. The elicited antibodies showed high specificity toward S100A9 without cross-reactivity toward S100A8, another member of the S100A family. When tested in metastatic mouse models of breast cancer and melanoma, the vaccines significantly reduced lung tumor nodules after intravenous challenge or postsurgical removal of the primary tumor. Mechanistically, the vaccines reduce the levels of S100A9 within the lungs and sera, thereby increasing the expression of immunostimulatory cytokines with antitumor function [(interleukin) IL-12 and interferonγ] while reducing levels of immunosuppressive cytokines (IL-10 and transforming growth factorβ). This also correlated with decreased myeloid-derived suppressor cell populations within the lungs. This work has wide-ranging impact, as S100A9 is overexpressed in multiple cancers and linked with poor prognosis in cancer patients. The data presented lay the foundation for the development of therapies and vaccines targeting S100A9 to prevent metastasis.

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.

Protein glycosylation has a huge impact on biological processes in all domains of life. The type of glycan present on a recombinant glycoprotein depends on protein intrinsic features and the glycosylation repertoire of the cell type used for expression. Glycoengineering approaches are used to eliminate unwanted glycan modifications and to facilitate the coordinated expression of glycosylation enzymes or whole metabolic pathways to furnish glycans with distinct modifications. The formation of tailored glycans enables structure-function studies and optimization of therapeutic proteins used in different applications. While recombinant proteins or proteins from natural sources can be in vitro glycoengineered using glycosyltransferases or chemoenzymatic synthesis, many approaches use genetic engineering involving the elimination of endogenous genes and introduction of heterologous genes to cell-based production systems. Plant glycoengineering enables the in planta production of recombinant glycoproteins with human or animal-type glycans that resemble natural glycosylation or contain novel glycan structures. This review summarizes key achievements in glycoengineering of plants and highlights current developments aiming to make plants more suitable for the production of a diverse range of recombinant glycoproteins for innovative therapies.

The world population's growing demand for food is expected to increase dramatically by 2050. The agronomic productivity for food is severely affected due to biotic and abiotic constraints. At a global level, insect pests alone account for ~20% loss in crop yield every year. Deployment of noxious chemical pesticides to control insect pests always has a threatening effect on human health and environmental sustainability. Consequently, this necessitates for the establishment of innovative, environmentally friendly, cost-effective, and alternative means to mitigate insect pest management strategies. According to a recent study, using chloroplasts engineered with double-strand RNA (dsRNA) is novel successful combinatorial strategy deployed to effectively control the most vexing pest, the western flower thrips (WFT: Frankliniella occidentalis). Such biotechnological avenues allowed us to recapitulate the recent progress of research methods, such as RNAi, CRISPR/Cas, mini chromosomes, and RNA-binding proteins with plastid engineering for a plausible approach to effectively mitigate agronomic insect pests. We further discussed the significance of the maternal inheritance of the chloroplast, which is the major advantage of chloroplast genome engineering.

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCVPA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCVPA and the wild-type subgroup 3 tobamovirus. TVCVPA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCVPA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCVPA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta2O5 sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCVPA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes.