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Abstract

Influenza virus is a globally important respiratory pathogen that causes a high degree of annual morbidity and mortality. Significant antigenic drift results in emergence of new, potentially pandemic, virus variants. The best prophylactic option for controlling emerging virus strains is to manufacture and administer pandemic vaccines in sufficient quantities and to do so in a timely manner without impacting the regular seasonal influenza vaccine capacity. Current, egg-based, influenza vaccine production is well established and provides an effective product, but has limited capacity and speed. To satisfy the additional global demand for emerging influenza vaccines, high-performance cost-effective technologies need to be developed. Plants have a potential as an economic and efficient large-scale production platform for vaccine antigens. In this study, a plant virus-based transient expression system was used to produce hemagglutinin (HA) proteins from the three vaccine strains used during the 2008-2009 influenza season, A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), and B/Florida/4/06, as well as from the recently emerged novel H1N1 influenza A virus, A/California/04/09. The recombinant plant-based HA proteins were engineered and produced in Nicotiana benthamiana plants within 2 months of obtaining the genetic sequences specific to each virus strain. These antigens expressed at the rate of 400-1300 mg/kg of fresh leaf tissue, with >70% solubility. Immunization of mice with these HA antigens induced serum anti-HA IgG and hemagglutination inhibition antibody responses at the levels considered protective against these virus infections. These results demonstrate the feasibility of our transient plant expression system for the rapid production of influenza vaccine antigens.

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... Their findings demonstrate the potential of the plant-produced H6N2 HA vaccine for poultry. HA from various influenza strains was expressed in plants, and its immunogenicity was assessed [80][81][82][83][84][85][86][87][88][89]. Modifications of the HA structure were made to achieve a high level of recombinant protein accumulation: the sequences were optimized, the transmembrane domain and native signal peptide were removed, and an endoplasmic retention signal was inserted at the C terminus. ...
... The yield of the transiently expressed protein was HA variant dependent. The yield of H3 was 200 mg/kg of fresh weight (FW) tobacco leaves [81], while the yield of H1 was 400-1300 mg/kg FW [82]. Shoji et al. generated trimeric HA, which mimics the authentic HA structure, by introducing a trimerization motif from a heterologous protein into the HA sequence [83]. ...
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Emerging and re-emerging zoonotic diseases cause serious illness with billions of cases, and millions of deaths. The most effective way to restrict the spread of zoonotic viruses among hu-mans and animals and prevent disease is vaccination. Recombinant proteins produced in plants offer an alternative approach for the development of safe, effective, inexpensive candidate vac-cines. Current strategies are focused on the production of highly immunogenic structural pro-teins, which mimic the organizations of the native virion but lack the viral genetic material. These include chimeric viral peptides, subunit virus proteins, and virus-like particles (VLPs). The latter, with their ability to self-assemble and thus resemble the form of virus particles, are gaining traction among plant-based candidate vaccines against many infectious diseases. In this review, we summarized the main zoonotic diseases and followed the progress in using plant expression systems for the production of recombinant proteins and VLPs used in the development of plant-based vaccines against zoonotic viruses.
... Plant expression systems have several potential benefits over conventional expression platforms. Recent studies have demonstrated the reliability of the plant expression system for the manufacture of highly valuable proteins, including Norwalk virus capsid protein , hemagglutinin of influenza virus (Shoji et al., 2012), norovirus Narita 104 virus-like particles (Mathew, Herbst-Kralovetz, & Mason, 2014), antibody 6D8 against Ebola virus , and consensus domain III of dengue virus E glycoprotein (Kim, Jang, Yang, & Kim, 2015). Plants are a very attractive platform because of their high level of scalability, low growth costs, ability to synthesize complex proteins, low risk of contamination with human pathogens, and potential for rapid production timescales (Burnett & Burnett, 2020). ...
... Many types of recombinant proteins, such as pharmaceuticals, vaccines, cytokines, and growth regulators, are produced in tobacco, Nicotiana benthamiana, and N. tabacum, because of the high levels of protein production with transient expression and easy manipulation. Recombinant proteins can be expressed in plants using transient expression, mediated by infiltration with Agrobacterium tumefaciens (called agroinfiltration), within $8 weeks after obtaining the corresponding DNA sequence (Shoji et al., 2012). This rapid production allows quick responses to epidemic or pandemic threats, as shown by the production of an anti-Ebola antibody cocktail, ZMapp (Qiu et al., 2014). ...
Chapter
Plants become a promising biofactory for the large-scale production of recombinant proteins due to low cost, scalability, and safety. Agroinfiltration of plant leaves with a plant viral vector carrying a gene of interest is a rapid and efficient method for protein production in plants. Currently this method is in use for producing a wide range of proteins for multiple applications, including vaccine antigens, antibodies, and protein nanoparticles such as virus-like particles. A number of pharmaceutical proteins produced by transient expression are currently in clinical development. Here, we describe potato virus X based vector pEff-GFP enabling fast and high-level expression of recombinant proteins in Nicotiana benthamiana plants. The pEff vector provides green fluorescent protein expression levels of up to 30% of total soluble protein (about 1 mg per g of fresh leaf tissue) and was successfully applied for the production of the immunogens of potential clinical interest.
... Moreover, the manufacturing of highly infectious pandemic virus particles requires high-level safety measurement; however, VLP production does not require any costly safety measurement since VLPs are composed of only non-infectious recombinant proteins. The VLP platform has been deployed to develop vaccines for the recurring influenza pandemic [29,[106][107][108]. The fast mass production of plant factoryderived VLPs could be a promising vaccine-manufacturing platform to cope with the COVID-19 pandemic. ...
... The company has more vaccine candidates in the production pipeline, including one against rotavirus and influenza; both are in phase 1 clinical trials [118]. Furthermore, it has produced a fully formulated HA VLP vaccine within 3 weeks of the release of the genetic sequence for the A/H1N1(A/California/04/09) strain [107]. These findings prove the capacity of the plant-based platform for rapid vaccine production, one of the most important aspects of vaccine development at the time of a pandemic. ...
Article
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The prevalence of the coronavirus disease 2019 (COVID-19) pandemic in its second year has led to massive global human and economic losses. The high transmission rate and the emergence of diverse SARS-CoV-2 variants demand rapid and effective approaches to preventing the spread, diagnosing on time, and treating affected people. Several COVID-19 vaccines are being developed using different production systems, including plants, which promises the production of cheap, safe, stable, and effective vaccines. The potential of a plant-based system for rapid production at a commercial scale and for a quick response to an infectious disease outbreak has been demonstrated by the marketing of carrot-cell-produced taliglucerase alfa (Elelyso) for Gaucher disease and tobacco-produced monoclonal antibodies (ZMapp) for the 2014 Ebola outbreak. Currently, two plant-based COVID-19 vaccine candidates, coronavirus virus-like particle (CoVLP) and Kentucky Bioprocessing (KBP)-201, are in clinical trials, and many more are in the preclinical stage. Interim phase 2 clinical trial results have revealed the high safety and efficacy of the CoVLP vaccine, with 10 times more neutralizing antibody responses compared to those present in a convalescent patient’s plasma. The clinical trial of the CoVLP vaccine could be concluded by the end of 2021, and the vaccine could be available for public immunization thereafter. This review encapsulates the efforts made in plant-based COVID-19 vaccine development, the strategies and technologies implemented, and the progress accomplished in clinical trials and preclinical studies so far.
... Plants are promising as a platform for the manufacture of influenza vaccines due to their scalability and the rapid production facilitated by transient expression. Whereas 6 months or more is needed to prepare sufficient quantities of egg-derived vaccine to meet global demand, the same can be achieved in plants within a few weeks (Shoji et al., 2012), as proven in the abovementioned DARPA Blue Angel program that produced 10 million doses in one month (Lomonossoff and D'Aoust, 2016). For this reason, although some influenza antigens have been produced in transgenic plants (Ceballo et al., 2017;Firsov et al., 2015;Lee et al., 2015), most have been transiently expressed in N. benthamiana (Hodgins et al., 2017;2019a,b;Landry et al., 2014;Shoji et al., 2009a,b;Shoji et al., 2013Shoji et al., , 2015 Table S1) including all vaccine candidates intended for commercial development. ...
... Various formats have been tested, including monomeric and trimeric HA subunits, but the most popular approach is the presentation of HA on the surface of VLPs. Typical VLPs require a viral coat protein component to form the core of the particle, and influenza vaccines based on tobacco mosaic virus have been developed using this principle by Fraunhofer CMB/iBio (Shoji et al., 2011(Shoji et al., , 2012. These systems can achieve high product yields: for example, vaccines against strains H3N2, H5N1 and H1N1 have been produced at yields of 50-200 mg/kg fresh leaf biomass (Shoji et al., 2008(Shoji et al., , 2011. ...
Article
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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 programs, 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, focusing on recent outbreaks of high‐mortality coronavirus infections and diseases that disproportionately affect the developing world.
... With product accumulation in the range of 0.1-4.0 g kg −1 biomass (Sainsbury and Lomonossoff, 2008;Zischewski et al., 2015;Yamamoto et al., 2018), larger-scale quantities (several grams) can be supplied after 4-8 weeks (Shoji et al., 2012), making this approach ideal for emergency responses to sudden disease outbreaks. Potential bottlenecks include the preparation of sufficiently large candidate libraries, ideally in an automated manner as described for conventional expression systems, and the infiltration of plants with a large number (>100) of candidates. ...
... 18 October 2020 | Volume 11 | Article 594019 FIGURE 2 | Comparison of mammalian cell culture and transient expression in plants for the production of emergency biopharmaceuticals. Timelines for conventional scheduling (black arrows) and accelerated procedures (double red arrows) are based on recent publications and announcements, as well as the authors' experience (Shoji et al., 2011(Shoji et al., , 2012Kelley, 2020). Transient expression allows much quicker vector development, process development, and reference material production, whereas the duration of toxicity studies is not reduced to the same degree because the time needed to run the studies remains the same regardless of the platform. ...
Article
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Several epidemic and pandemic diseases have emerged over the last 20 years with increasing reach and severity. The current COVID-19 pandemic has affected most of the world's population, causing millions of infections, hundreds of thousands of deaths, and economic disruption on a vast scale. The increasing number of casualties underlines an urgent need for the rapid delivery of therapeutics, prophylactics such as vaccines, and diagnostic reagents. Here, we review the potential of molecular farming in plants from a manufacturing perspective, focusing on the speed, capacity, safety, and potential costs of transient expression systems. We highlight current limitations in terms of the regulatory framework, as well as future opportunities to establish plant molecular farming as a global, decentralized emergency response platform for the rapid production of biopharmaceuticals. The implications of public health emergencies on process design and costs, regulatory approval, and production speed and scale compared to conventional manufacturing platforms based on mammalian cell culture are discussed as a forward-looking strategy for future pandemic responses.
... Recently, Medicago (Quebec, Canada), Kentucky BioProcessing (Owensboro, KT, USA), and iBio (Bryan, TX, USA) joined the global race for developing potential plant-based vaccines for COVID-19 [126]. By using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than 4 weeks after receiving the corresponding gene construct [5,59,127]. ...
... Recently, Medicago (Quebec, Canada), Kentucky BioProcessing (Owensboro, KT, USA), and iBio (Bryan, TX, USA) joined the global race for developing potential plant-based vaccines for COVID-19 [126]. By using the transient expression platform, recombinant protein production in plants could be scaled up rapidly, and milligram quantities of proteins could be produced in a timeframe of less than 4 weeks after receiving the corresponding gene construct [5,59,127]. Alternately, chloroplast expression focuses on expressing the transgenes in chloroplast by the precise insertion of foreign DNA by homologous recombination into the chloroplast genome. ...
Article
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The demand for recombinant proteins in terms of quality, quantity, and diversity is increasing steadily, which is attracting global attention for the development of new recombinant protein production technologies and the engineering of conventional established expression systems based on bacteria or mammalian cell cultures. Since the advancements of plant genetic engineering in the 1980s, plants have been used for the production of economically valuable, biologically active non-native proteins or biopharmaceuticals, the concept termed as plant molecular farming (PMF). PMF is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. The development and improvement of the transient expression system has significantly reduced the protein production timeline and greatly improved the protein yield in plants. The major factors that drive the plant-based platform towards potential competitors for the conventional expression system are cost-effectiveness, scalability, flexibility, versatility, and robustness of the system. Many biopharmaceuticals including recombinant vaccine antigens, monoclonal antibodies, and other commercially viable proteins are produced in plants, some of which are in the pre-clinical and clinical pipeline. In this review, we consider the importance of a plant- based production system for recombinant protein production, and its potential to produce biopharmaceuticals is discussed.
... The Fraunhofer Centre for Molecular Biotechnology (USA) has described the development of a similar transient plant expression platform for the production of soluble HA antigens which has been reported to yield purified vaccines in just over a month (Shoji et al., 2012). Both of these platforms are highly flexible and have been used to produce HA from several seasonal and pandemic influenza subtypes (D'Aoust et al., 2008;Mett et al., 2008;Pillet et al., 2015;Shoji et al., 2008Shoji et al., , 2009aShoji et al., ,b, 2011Shoji et al., , 2012. ...
... The Fraunhofer Centre for Molecular Biotechnology (USA) has described the development of a similar transient plant expression platform for the production of soluble HA antigens which has been reported to yield purified vaccines in just over a month (Shoji et al., 2012). Both of these platforms are highly flexible and have been used to produce HA from several seasonal and pandemic influenza subtypes (D'Aoust et al., 2008;Mett et al., 2008;Pillet et al., 2015;Shoji et al., 2008Shoji et al., , 2009aShoji et al., ,b, 2011Shoji et al., , 2012. These antigens have demonstrated promising immunogenicity in preclinical animal models with several reports of protection against lethal virus challenge (D' Aoust et al., 2008;Mett et al., 2008;Pillet et al., 2015;Shoji et al., 2008Shoji et al., , 2009aShoji et al., ,b, 2011. ...
Article
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Plant molecular farming offers a cost effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs‐of‐concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date the only plant‐derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post‐translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review we will address potential shortcomings of plant‐expression systems and discuss strategies to optimally exploit the technology for the production of immunologically‐relevant and structurally authentic glycoproteins for use as vaccine immunogens. This article is protected by copyright. All rights reserved.
... Biopharming, or plant molecular farming, refers to the use of genetic tools to produce a wide range of pharmaceuticals. Plants have already been used to produce antibodies and vaccines for humans, animals, and aquaculture (Shoji et al., 2012;Takeyama et al., 2015;Yao et al., 2015;Lefebvre and Lécuyer, 2017;Zahara et al., 2017;Su et al., 2021). Recently, plants have been explored as a rapid alternative biofactory for the production of COVID vaccines through the expression of Virus-like particles exposing an immunogenic part of the Spike S protein (Dhama et al., 2020;Maharjan and Choe, 2021). ...
Article
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Mechanisms devoted to the secretion of proteins via extracellular vesicles (EVs) have been found in mammals, yeasts, and plants. Since they transport a number of leader-less proteins to the plasma membrane or the extracellular space, EVs are considered part of Unconventional protein secretion (UPS) routes. UPS involving EVs are a relatively new field in plants. Aside from their role in plant physiology and immunity, plant extracts containing EVs have also been shown to be beneficial for human health. Therefore, exploring the use of plant EVs in biomedicine and their potential as drug delivery tools is an exciting avenue. Here we give a summary of the state of knowledge on plant EVs, their crosstalk with mammalian systems and potential research routes that could lead to practical applications in therapeutic drug delivery.
... In a study conducted in rats in 2012, transgenic tobacco plants producing HPAIV H5N1 from the avian flu virus offered a boost to IgG stimulation. 17,25,26 Transgenic tobacco plants that express a protein from Eimeriatenella, the agent that causes coccidiosis, as well as transgenic tobacco plants that combat anthrax, have recently been identified. In the latter, the tobacco produced a shielding antigen (PA), which resulted in increased blood IgA and IgG levels in mice. ...
Article
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A New approach for vaccine delivery Antigen is the use of inexpensive of oral vaccines. Is ideal for edible vaccination since it grows quickly and has a lot of nutrients that improve the immune system. Edible oral vaccines supply exciting Prospects for considerably reducing the burden of disease like hepatitis and diarrhea Significantly within the developing world wherever storing and Administering vaccines are typically major problem. They have several benefits as they trigger the Immunity at the tissue layer surface which is the body’s first line of defense. Painful vaccination processes are replaced by an edible vaccine. In comparison to traditional vaccines, edible vaccines are needle-free, low-cost, do not require refrigeration, may be stored close to the point of application, are safe, and give mucosal and systemic protection. Vaccines play a vital role in the prevention and treatment of a wide range of diseases. The Review's major goal is to provide comprehensive information on edible vaccines. The main aim of the Review is to provide an all over information related to edible vaccine. The hope is the edible vaccine could be grown in the developing Many of countries Where their need is more. Keywords: edible vaccine, fruits, immunity, vaccination, transgene, antigen
... This method is of particular interest when easy access to the roots is needed or when the artificial substrates used for soilless culture are banned to prevent them from impacting the environment through their disposal or production [15,16]. Noteworthily, HPA-based production of plant-specialized metabolites has found successful applications in the cosmetic and pharmaceutic industries with, for example, the production of prenylated polyphenols [17] and plant-based vaccines [18]. Several studies on the soilless cultivation of medicinal and aromatic crops have demonstrated advantages in terms of biomass yield and the quality of active chemical compounds [19][20][21]. ...
Article
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Vetiver (Chrysopogon zizanioides (L.) Roberty) is a major tropical perfume crop. Access to its essential oil (EO)-filled roots is nevertheless cumbersome and land-damaging. This study, therefore, evaluated the potential of vetiver cultivation under soilless high-pressure aeroponics (HPA) for volatile organic compound (VOC) production. The VOC accumulation in the roots was investigated by transmission electron microscopy, and the composition of these VOCs was analyzed by gas chromatography coupled with mass spectrometry (GC/MS) after sampling by headspace solid-phase microextraction (HS-SPME). The HPA-grown plants were compared to plants that had been grown in potting soil and under axenic conditions. The HPA-grown plants were stunted, demonstrating less root biomass than the plants that had been grown in potting soil. The roots were slender, thinner, more tapered, and lacked the typical vetiver fragrance. HPA cultivation massively impaired the accumulation of the less-volatile hydrocarbon and oxygenated sesquiterpenes that normally form most of the VOCs. The axenic, tissue-cultured plants followed a similar and more exacerbated trend. Ultrastructural analyses revealed that the HPA conditions altered root ontogeny, whereby the roots contained fewer EO-accumulating cells and hosted fewer and more immature intracellular EO droplets. These preliminary results allowed to conclude that HPA-cultivated vetiver suffers from altered development and root ontology disorders that prevent EO accumulation.
... One advantage of plant-based expression systems is biosafety due to lack of contamination with animal-borne viruses. Furthermore, there are technical advantages associated with up-stream processing: For instance, a rapid scale-up of production using Nicotiana benthamiana and a transient expression system is feasible within 2 months (Shoji et al., 2012;Capell et al., 2020), which would allow a quick response to address a public health crisis. During the current COVID-19 pandemic, it became evident that diagnostic reagents and vaccine production capacity was not sufficient to meet demand, and transient expression in plants could compensate this (Tusé et al., 2020). ...
Article
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The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO 2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 µM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-kynurenine (100 µM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 h after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first 2 hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.
... One advantage of plant-based expression systems is biosafety due to lack of contamination with animal-borne viruses. Furthermore, there are technical advantages associated with up-stream processing: For instance, a rapid scale-up of production using Nicotiana benthamiana and a transient expression system is feasible within two months (Shoji et al., 2012;Capell et al., 2020), which would allow a quick response to address a public health crisis. During the current COVID-19 pandemic, it became evident that diagnostic reagents and vaccine production capacity was not sufficient to meet demand, and transient expression in plants could compensate this (Tusé et al., 2020). ...
Preprint
Full-text available
The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 µM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-Kynurenine (100 µM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 hours after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first two hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was .
... Furthermore, the cost of purifying the protein of interest and the cost for testing virus-free are reduced in plant expression system. By using A. tumefaciens and/or viral vector-mediated transient expression, recombinant protein expression in plants can be achieved approximately 8 weeks after obtaining the corresponding DNA sequence (Gleba et al. 2014;Shoji et al. 2012). In addition, plant expression systems have the advantage of producing intrinsically disordered proteins, including the anti-cancer mistletoe lectin viscumin (Gengenbach et al. 2019), which are not synthesized efficiently in mammalian cells or prokaryotes due to their toxicity or complex structure, respectively. ...
Article
The production of recombinant proteins is important in academic research to identify protein functions. Moreover, recombinant enzymes are used in the food and chemical industries, and high-quality proteins are required for diagnostic, therapeutic, and pharmaceutical applications. Though many recombinant proteins are produced by microbial or mammalian cell-based expression systems, plants have been promoted as alternative, cost-effective, scalable, safe, and sustainable expression systems. The development and improvement of transient expression systems have significantly reduced the period of protein production and increased the yield of recombinant proteins in plants. In this review, we consider the importance of plant-based expression systems for recombinant protein production and as genetic engineering tools.
... Two important plant-based expression strategies (transient and transgenic) minimize the production cost compared to others. The transient strategy in plants via vacuum infiltration with Agrobacterium tumefaciens transformed with a plant expression vector harboring desired protein encoding gene [6]. However, this technique is fast but requires continuous infiltration of transformed agrobacterium, coinciding with the same production cost as fermentation. ...
Article
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Plants are becoming useful platforms for recombinant protein production at present time. With the advancement of efficient molecular tools of genomics, proteomics, plants are now being used as a biofactory for production of different life saving therapeutics. Plant-based biofactory is an established production system with the benefits of cost-effectiveness, high scalability, rapid production, enabling post-translational modification, and being devoid of harmful pathogens contamination. This review introduces the main challenges faced by plant expression system: post-translational modifications, protein stability, biosafety concern and regulation. It also summarizes essential factors to be considered in engineering plants, including plant expression system, promoter, post-translational modification, codon optimization, and fusion tags, protein stabilization and purification, subcellular targeting, and making vaccines in an edible way. This review will be beneficial and informative to scholars and readers in the field of plant biotechnology.
... Another study reported enhanced immunogenicity of recombinant HA in an enveloped VLP over soluble antigen [91]. 400-1300 mg protein obtained from 1 Kg of fresh infiltrated leaf tissue [92]. Another study reported good immunogenicity and safety profiles of HAC1 and HAI-05 in animal pre-clinical studies [93]. ...
Article
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Many pathogenic viral pandemics have caused threats to global health; the COVID-19 pandemic is the latest. Its transmission is growing exponentially all around the globe, putting constraints on the health system worldwide. A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), causes this pandemic. Many candidate vaccines are available at this time for COVID-19, and there is a massive international race underway to procure as many vaccines as possible for each country. However, due to heavy global demand, there are strains in global vaccine production. The use of a plant biotechnology-based expression system for vaccine production also represents one part of this international effort, which is to develop plant-based heterologous expression systems, virus-like particles (VLPs)-vaccines, antiviral drugs, and a rapid supply of antigen-antibodies for detecting kits and plant origin bioactive compounds that boost the immunity and provide tolerance to fight against the virus infection. This review will look at the plant biotechnology platform that can provide the best fight against this global pandemic.
... Industry has also been discouraged by the timeframe of 6-18 months needed to regenerate stable, transgenic plants and the bottlenecks along the path to regulatory approval Tusé et al. 2020). Furthermore, the highest level of product accumulation recorded in plants and plant cells is currently * 4 g kg -1 for GFP and similar levels have been achieved for monoclonal antibodies and influenza antigens as well (Yamamoto et al. 2018;Shoji et al. 2012;Zischewski et al. 2015) whereas mammalian cells often achieve yields [ 25 g L -1 with well-characterized products such as antibodies (Yang et al. 2016). Product yields in plants can also vary substantially within the biomass Buyel and Fischer 2012;Knödler et al. 2019), especially if host reactions, such as the response to infiltrating bacteria during transient expression, lead to the activation of endogenous proteases (Grosse-Holz et al. 2017). ...
Article
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Plants have provided humans with useful products since antiquity, but in the last 30 years they have also been developed as production platforms for small molecules and recombinant proteins. This initially niche area has blossomed with the growth of the global bioeconomy, and now includes chemical building blocks, polymers and renewable energy. All these applications can be described as “plant molecular farming” (PMF). Despite its potential to increase the sustainability of biologics manufacturing, PMF has yet to be embraced broadly by industry. This reflects a combination of regulatory uncertainty, limited information on process cost structures, and the absence of trained staff and suitable manufacturing capacity. However, the limited adaptation of plants and plant cells to the requirements of industry-scale manufacturing is an equally important hurdle. For example, the targeted genetic manipulation of yeast has been common practice since the 1980s, whereas reliable site-directed mutagenesis in most plants has only become available with the advent of CRISPR/Cas9 and similar genome editing technologies since around 2010. Here we summarize the applications of new genetic engineering technologies to improve plants as biomanufacturing platforms. We start by identifying current bottlenecks in manufacturing, then illustrate the progress that has already been made and discuss the potential for improvement at the molecular, cellular and organism levels. We discuss the effects of metabolic optimization, adaptation of the endomembrane system, modified glycosylation profiles, programmable growth and senescence, protease inactivation, and the expression of enzymes that promote biodegradation. We outline strategies to achieve these modifications by targeted gene modification, considering case-by-case examples of individual improvements and the combined modifications needed to generate a new general-purpose “chassis” for PMF.
... Plants offer huge advantages compared with platforms based on traditional mammalian cell cultures. Using a transient expression system mediated by agroinfiltration and/or viral vectors [3,4], recombinant protein engineering and production in transgenic plants can be possible within 2 months after receiving the corresponding RNA sequence [5]. This speedy production system can rapidly address any pandemic crisis like COVID-19, as impressively shown by the production of an antibody cocktail for Ebola virus disease [6]. ...
Article
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The emergence of the COVID-19 pandemic has led to significant public health crisis all over the world. The rapid spreading nature and high mortality rate of COVID-19 places a huge pressure on scientists to develop effective diagnostics and therapeutics to control the pandemic. Some scientists working on plant biotechnology together with commercial enterprises for the emergency manufacturing of diagnostics and therapeutics have aimed to fulfill the rapid demand for SARS-CoV-2 protein antigen and antibody through a rapid, scalable technology known as transient/stable expression in plants. Plant biotechnology using transient/stable expression offers a rapid solution to address this crisis through the production of low-cost diagnostics, antiviral drugs, immunotherapy, and vaccines. Transient/stable expression technology for manufacturing plant-based biopharmaceuticals is already established at commercial scale. Here, current opinions regarding how plant biotechnology can help fight against COVID-19 through the production of low-cost diagnostics and therapeutics are discussed.
... This mapping can include leaf color or absorbance data that can indicate malnutrition or the onset of infections, respectively. Importantly, these monitoring techniques are contact-free, non-invasive and non-destructive, allowing repeated measurements during a production campaign that can last the 5-9 weeks required for tobacco and N. benthamiana (Shoji et al., 2012), minimizing the risk of unintended contamination with pathogens. Furthermore, monitoring individual plants can provide new insights into the USP of plant molecular farming. ...
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The market for biopharmaceuticals is dominated by recombinant proteins and is driven mainly by the development of vaccines and antibodies. Manufacturing predominantly relies on fermentation-based production platforms, which have limited scalability and suffer from high upstream process costs. As an alternative, the production of recombinant proteins in whole plants (plant molecular farming) provides a scalable and cost efficient upstream process because each plant functions as a self-contained bioreactor, avoiding costs associated with single-use devices and cleaning-in-place. Despite many proof-of-concept studies and the approval of a few products as medical devices, the only approved pharmaceutical proteins manufactured in whole plants have been authorized under emergency protocols. The absence of approvals under standard clinical development pathways in part reflects the lack of standardized process equipment and unit operations, leading to industry inertia based on familiarity with fermenter systems. Here we discuss the upstream production steps of plant molecular farming by transient expression in intact plants, including seeding, plant cultivation, infiltration with Agrobacterium tumefaciens, post-infiltration incubation, and harvesting. We focus on cultivation techniques because they strongly affect the subsequent steps and overall process design. We compare the benefits and drawbacks of open field, greenhouse and vertical farm strategies in terms of upfront investment costs, batch reproducibility, and decoupling from environmental impacts. We consider process automation, monitoring and adaptive process design in the context of Industry 4.0, which can boost process efficiency and batch-to-batch uniformity to improve regulatory compliance. Finally, we discuss the costs–benefit aspects of the different cultivation systems.
... Furthermore, plants do not support the growth of human pathogens, they can be engineered to carry out authentic post-translational modifications (Jansing et al., 2019), and they can accumulate proteins such as toxins that kill mammalian cells (Gamerith et al., 2017;Gengenbach et al., 2019). Transient expression in plants is also rapid, with even large-scale experiments requiring only weeks from gene to product (Garabagi et al., 2012;Shoji et al., 2012;Sainsbury and Lomonossoff, 2014). In contrast, the development of mammalian cell lines takes many months even before production commences. ...
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The high-throughput screening of recombinant protein expression is advantageous during early process development because it allows the identification of optimal expression constructs and process conditions. Simple screening platforms based on microtiter plates are available for microbes and animal cells, but this was not possible for plants until the development of plant cell packs (PCPs), also known as “cookies,” which provide a versatile and scalable screening tool for recombinant protein production. PCPs are prepared from plant cell suspension cultures by removing the medium and molding the biomass. PCPs can be cast into 96-well plates for high-throughput screening, but the manual handling effort currently limits the throughput to ∼500 samples per day. We have therefore integrated the PCP method with a fully automated laboratory liquid-handling station. The “robot cookies” can be prepared and infiltrated with Agrobacterium tumefaciens by centrifugation, minimizing operator handling and reducing the likelihood of errors during repeated runs, such as those required in a design of experiments approach. The accumulation of fluorescent protein in the cytosol, apoplast, endoplasmic reticulum or plastids is easily detected using an integrated plate reader, reducing the inter-experimental variation to <5%. We also developed a detergent-based chemical lysis method for protein extraction in a 96-well format, which was adapted for automated downstream processing using miniaturized columns allowing subsequent protein analysis. The new automated method reduces the costs of the platform to <0.5 € per PCP infiltration (a saving of >50%) and facilitates a five-fold increase in throughput to >2500 samples per day.
... When the rats were challenged with this antigen, then it stimulated the expression of IgG immunoglobuline. 36,72 Recently, researchers reported the expression of anthrax and Eimeria tenella antigen in tobacco plants. 21,69 ...
... HIV vaccine implementation will also require unprecedented scalability and the potential need for annually repeated immunizations, as already occurs with seasonal influenza vaccines, could eclipse all available manufacturing capacity (Mortimer et al., 2012). Recently, high yields and promising immunogenicity of plant-produced influenza virus haemagglutinin-derived antigens have been reported, several of which have advanced into clinical trials (D' Aoust et al., 2008;Shoji et al., 2008;Shoji et al., 2009a;Shoji et al., 2009b;Bosch and Schots 2010;Landry et al., 2010;Madhun et al., 2011;Chichester et al., 2012;Shoji et al., 2012;Cummings et al., 2014). Medicago Inc (USA) has also demonstrated the scalability of plant-based expression by producing 10 million doses of fully formulated influenza H1N1pdm vaccines in one month ( Yusibov et al., 2015). ...
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The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.
... While egg-based vaccine production takes at least 6 months, Medicago Inc. reported that the functional hemagglutinin (HA) virus like particle vaccine can be produced in plants within 21 days from identification of sequence for pandemic influenza [15]. The Fraunhofer Centre for Molecular Biotechnology also reported that it takes just over a month to yield purified HA antigens [16]. These vaccines have shown to be highly immunogenic in preclinical animal model, which showed protection against lethal virus challenge, and in human volunteers. ...
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Vaccination is one of the most successful strategies to prevent diseases caused by pathogens. Although various expression systems including Escherichia coli, yeast, insect, and mammalian cells are currently used for producing many of vaccines, these conventional platforms have the limitation of post-translational modification, high cost, and expensive scalability. In this respect, the plant-based expression system has been considered as an attractive platform to produce recombinant vaccines due to fast, cost-effective and scalable production as well as safety. This review discusses the development of plant-derived vaccines and the current stage of plant-based expression system.
... HA vaccine with varying glycosylation pattern could be produced in plant by genetic engineering of the genes involved in glycosylation pathway. Many vaccine antigens for the various strain and subtype of human (H1N1, H3N2) and avian influenza (H5N1) have been expressed transiently in N. benthamiana and advanced to the different phases of clinical trial [15][16][17][18]. However, there is no report on the canine influenza vaccine antigen expressed in plant. ...
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Purpose: Canine influenza virus (CIV), H3N2, carries potentiality for zoonotic transmission and genetic assortment which raises a concern on possible epidemics, and human threats in future. To manage possible threats, the development of rapid and effective methods of CIV vaccine production is required. The plant provides economical, safe, and robust production platform. We investigated whether hemagglutinin (HA) antigen from Korea-originated CIV could be produced in Nicotiana benthamiana and lettuce, Lactuca sativa by a DNA viral vector system. Materials and methods: We used DNA sequences of the HA gene from Korean CIV strain influenza A/canine/Korea/S3001/2015 (H3N2) for cloning into a geminiviral expression vectors to express recombinant HA (rHA) antigen in the plant. Agrobacterium-mediated infiltration was performed to introduce HA-carrying vector into host plants cells. Laboratory-grown N. benthamiana, and grocery-purchased or hydroponically-grown lettuce plant leaves were used as host plants. Results: CIV rHA antigen was successfully expressed in host plant species both N. benthamiana and L. sativa by geminiviral vector. Both complex-glycosylated and basal-glycosylated form of rHA were produced in lettuce, depending on presence of endoplasmic reticulum (ER) retention signal. In terms of rHA expression level, canine HA (H3N2) showed preference to the native signal peptide than ER retention signal peptide in the tested geminiviral vector system. Conclusion: Grocery-purchased lettuce leaves could serve as an instant host system for the transient expression of influenza antigen at the time of emergency. The geminiviral vector was able to induce expression of complex-glycosylated and basal-glycosylated rHA in lettuce and tobacco.
... Several studies in animal models have assessed plant-based candidate vaccines against seasonal, pandemic and potentially pandemic influenza strains, at different doses and with or without adjuvant. The results showed good immunogenicity (humoral and cellular) and safety profiles, underlining the great advantage of the very rapid development of a plant vaccine and raising the need to further investigate whether the assays used for the evaluation of this kind of vaccine are adequate [168,[172][173][174][175][176][177][178]. ...
Article
Introduction: Current influenza vaccines are mainly produced from viruses propagated in eggs, an established process that has been developed over 70 years. However, this technology presents some drawbacks and other platforms are under development or have been already developed in order to replace or to be used alongside the old one. Area covered: The present review provides an overview of influenza vaccine production, starting from egg-based technology, to cell-derived vaccines, until the novel platforms and technologies for the production of influenza vaccines such as DNA-based vaccines, virus-like particles and plant-based technology. Expert opinion: The ideal method of production should have certain characteristics such as great flexibility and scalability, the viruses should be representative of the circulating strains, process should be standardized and controlled, and it should be possible to start production as soon as the sequence of the new influenza strain is available. However, it is important not to underestimate the fact that some parts of the vaccine production process have been established for egg-based vaccines. Thus, changes in vaccine production methods are not merely “technical changes”; rather, they involve various aspects that slow down the authorization of new influenza vaccines and make the complete replacement of egg-based production unlikely in the near future.
... To date, there are some plant-based vaccines for the hepatitis B virus (HBV), rabies virus, Norwalk virus, enterotoxigenic E. coli, and Vibrio cholerae in phase 1 clinical trials (Table 1). Many others are still in preclinical development, including vaccines targeting a variety of pathogens such as avian influenza viruses (HPAI H5N1) [39], Helicobacter pylori [40], and coronaviruses [41]. ...
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Vaccines are recognized worldwide as one of the most important tools for combating infectious diseases. Despite the tremendous value conferred by currently available vaccines toward public health, the implementation of additional vaccine platforms is also of key importance. In fact, currently available vaccines possess shortcomings, such as inefficient triggering of a cell-mediated immune response and the lack of protective mucosal immunity. In this regard, recent work has been focused on vaccine delivery systems, as an alternative to injectable vaccines, to increase antigen stability and improve overall immunogenicity. In particular, novel strategies based on edible or intradermal vaccine formulations have been demonstrated to trigger both a systemic and mucosal immune response. These novel vaccination delivery systems offer several advantages over the injectable preparations including self-administration, reduced cost, stability, and elimination of a cold chain. In this review, the latest findings and accomplishments regarding edible and intradermal vaccines are described in the context of the system used for immunogen expression, their molecular features and capacity to induce a protective systemic and mucosal response.
... Even under more controlled conditions as found in a greenhouse (Ma et al., 2015;Sack et al., 2015) or vertical farm (Wirz et al., 2012;Holtz et al., 2015), multi-tonne scale production may still be possible (Buyel et al., 2017). A fourth advantage is that recombinant protein expression in plants can be achieved ∼8 weeks after receiving the corresponding DNA sequence (Shoji et al., 2012), typically using transient expression mediated by infiltration with Agrobacterium tumefaciens and/or viral vectors (Peyret and Lomonossoff, 2013;Gleba et al., 2014). This rapid production allows quick responses to epidemic or pandemic threats, as impressively shown by the production of an anti-Ebola antibody cocktail (Qiu et al., 2014). ...
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Plants have unique advantages over other systems such as mammalian cells for the production of valuable small molecules and proteins. The benefits cited most often include safety due to the absence of replicating human pathogens, simplicity because sterility is not required during production, scalability due to the potential for open-field cultivation with transgenic plants, and the speed of transient expression potentially providing gram quantities of product in less than 4 weeks. Initially there were also significant drawbacks, such as the need to clarify feed streams with a high particle burden and the large quantities of host cell proteins, but efficient clarification is now readily achieved. Several additional advantages have also emerged reflecting the fact that plants are essentially biodegradable, single-use bioreactors. This article will focus on the exploitation of this concept for the production of biopharmaceutical proteins, thus improving overall process economics. Specifically, we will discuss the single-use properties of plants, the sustainability of the production platform, and the commercial potential of different biomass side streams. We find that incorporating these side streams through rational process integration has the potential to more than double the revenue that can currently be achieved using plant-based production systems.
... When the rats were challenged with this antigen, then it stimulated the expression of IgG immunoglobuline. 36,72 Recently, researchers reported the expression of anthrax and Eimeria tenella antigen in tobacco plants. 21,69 ...
Chapter
Vaccines exhibit great potential to fight against infectious disease. Injected vaccines only pass mucous membranes due to which it generates a poor stimulation in mucosal immune responses. Edible vaccines make contact with the lining of digestive tract by which it activates the mucosal as well as systemic immunity. These properties of edible vaccines provide more effective protection from the dangerous microorganisms which cause deadly diseases. However the above great properties of edible vaccines there are many other issues which are still to be addressed. For example, the vaccine production from the plants is in very low amount. While solving such problems, the researchers also have to be sure that the vaccine produced by the fruits should have a predictable dose.
... An effective response to an emerging pandemic on a global scale will require a combination of technologies to enable sufficient vaccine supply, distribution, and effectiveness. Modern manufacturing approaches rely on producing defined antigens in recombinant systems including mammalian cells (1,2), Escherichia coli (3), baculovirus (4), and plants (5,6). ...
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Adjuvants are key to shaping the immune response to vaccination, but to date, no adjuvant suitable for human use has been developed for intradermal vaccines. These vaccines could be self-administered and sent through the mail as they do not require long needles or technical expertise in immunization. In the event of a pandemic outbreak, this approach could alleviate the congregation of patients in health centers and thus reduce the potential of these centers to enhance the spread of lethal infection. A reliable and potent vaccine system for self-administration would provide an effective countermeasure for delivery through existing product distribution infrastructure. We report results from preclinical and clinical trials that demonstrate the feasibility of an adjuvanted, intradermal vaccine that induced single shot protection in ferrets and seroprotection in humans against one of the more lethal strains of pandemic flu, Indonesia H5N1. In the human trial, the vaccine was safe and clinical responses were above approvable endpoints for a protective flu vaccine. Inclusion of a modern TLR4 (Toll-like receptor 4) agonist–based adjuvant was critical to the development of the response in the intradermal groups. In humans, this is the first report of a safe and effective intradermal adjuvant, GLA-AF (aqueous formulation of glucopyranosyl lipid adjuvant), and provides a future path for developing a vaccine-device combination for distribution by mail and self-administration in case of a pandemic.
... We speculated that such negative effects would not occur in plant cells because they do not possess a PHACTR1 gene, and the equivalent regulatory cascade of Rho family of GTPases [16], which control PHACTR1 subcellular localization [15], are only distantly related to their human counterparts. Transient expression in plants is advantageous because the gene-to-product cycle takes ∼4 weeks [17], which allows the rapid screening of PHACTR1 variants. Plants can also support very-large scale production when bulk quantities of protein are required, e.g. for therapeutic purposes [18]. ...
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Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones. We therefore used a design of experiments approach to test the suitability of three plant-based systems for the expression of satisfactory quantities of recombinant PHACTR1, namely transient expression in tobacco (Nicotiana tabacum) BY-2 plant cell packs (PCPs), whole N. benthamiana leaves and BY-2 cell lysate (BYL). The highest yield was achieved using the BYL: up to 120 mg product kg-1 biomass equivalent within 48 h of translation. This was 1.3-fold higher than transient expression in N. benthamiana together with the silencing inhibitor p19, and 6-fold higher than the PCP system. The presence of Triton X-100 in the extraction buffer increased the recovery of PHACTR1 by 2-200-fold depending on the conditions. PHACTR1 was incompatible with biomass blanching and was stable for less than 16 h in raw plant extracts. Purification using a DDK-tag proved inefficient whereas 15% purity was achieved by immobilized metal affinity chromatography.
... Gómez et al. [111] endeavored to more effectively express the virus antigen in transgenic tobacco. In 2012, transgenic tobacco plants expressing HPAIV H5N1 from avian flu virus gave rise to IgG stimulation when tested in rats [112,113]. Recently, transgenic tobacco plants expressing a protein from Eimeria tenella, the agent that causes coccidiosis [84], and transgenic tobacco plants to combat anthrax [114] were reported. In the latter, the tobacco expressed a protective antigen (PA) that resulted in elevated serum IgA and IgG in murine models. ...
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The lethality of infectious diseases has decreased due to the implementation of crucial sanitary procedures such as vaccination. However, the resurgence of pathogenic diseases in different parts of the world has revealed the importance of identifying novel, rapid, and concrete solutions for control and prevention. Edible vaccines pose an interesting alternative that could overcome some of the constraints of traditional vaccines. The term “edible vaccine” refers to the use of edible parts of a plant that has been genetically modified to produce specific components of a particular pathogen to generate protection against a disease. The aim of this review is to present and critically examine “edible vaccines” as an option for global immunization against pathogenic diseases and their outbreaks and to discuss the necessary steps for their production and control and the list of plants that may already be used as edible vaccines. Additionally, this review discusses the required standards and ethical regulations as well as the advantages and disadvantages associated with this powerful biotechnology tool.
... In contrast, there are two major expression strategies using intact plants that have the potential to reduce the operating costs for upstream production substantially compared to cell cultures. In the transient expression approach, wild-type plants are vacuum infiltrated with Agrobacterium tumefaciens carrying a vector encoding the mAb heavy and light chains, allowing large quantities of mAb to accumulate within 5-8 days (Shoji et al., 2012). Although this method is rapid, its potential drawback for VLS production is that each plant in every batch must be infiltrated with bacteria, which merely transfers the fermentation costs to the production of bacteria (Houdelet et al., 2017). ...
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Gene technology has facilitated the biologization of manufacturing, i.e. the use and production of complex biological molecules and systems at an industrial scale. Monoclonal antibodies (mAbs) are currently the major class of biopharmaceutical products, but they are typically used to treat specific diseases which individually have comparably low incidences. The therapeutic potential of mAbs could also be used for more prevalent diseases, but this would require a massive increase in production capacity that could not be met by traditional fermenter systems. Here we outline the potential of plants to be used for the very-large-scale (VLS) production of biopharmaceutical proteins such as mAbs. We discuss the potential market sizes and their corresponding production capacities. We then consider available process technologies and scale-down models and how these can be used to develop VLS processes. Finally, we discuss which adaptations will likely be required for VLS production, lessons learned from existing cell culture-based processes and the food industry, and practical requirements for the implementation of a VLS process.
... A transgenic plant (Arabidopsis thaliana) containing HPAIV H5N1 antigen in its endoplasmic reticulum, which can be used as edible vaccine or as diagnostic reagents for avian influenza virus infection, was developed by Hwang et al., (2012). Shoji et al., 2012 reported that HA proteins were expressed on tobacco plant and oral immunization of mice with these HA antigens induced serum antihemagglutinin IgG. Hemagglutination inhibiting antibody confers protection against subsequent infections. ...
Article
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Plant based edible vaccines are one of the novel branches of vaccinology. Candidate antigen can be expressed on selected plant species through various biotechnological approaches. Stable integration of selected antigen into plant genome can be achieved through vector mediated or biolistic method of transgenesis. Another method is transient expression of candidate antigen using agroinfiltration or infection with modified RNA viruses. These types of vaccines have been developed against poultry diseases also. Immunogenic proteins of avibirnavirus (VP2), avian reo virus (σC), Newcastle disease virus (HN, F), avian coronavirus (S1), avian influenza virus (rHA0), chicken infectious anaemia virus (VP1) and Eimeria tenella (EtMIC2) were expressed in selected plants. Oral immunization with these transgenic plants followed by challenge with infectious organisms exhibited protective efficacy. Edible vaccines against poultry diseases are cost effective, thermostable and devoid of human or animal pathogens and microbial toxins. Other points in credit for edible vaccines include suitability for mass vaccination, ease of administration, storage stability, least stress, needle free delivery, no muscle damage etc. Commercial preparations of plant based edible vaccines are likely become a reality in near future. Before that, problems like standardization of expressed antigen concentration, vaccine composition, vaccine efficacy, safety and stability under field conditions have to be looked into. Plant based vaccines against various poultry diseases may become an alternative to conventional vaccination programmes in coming decades.
... In contrast, there are two major expression strategies using intact plants that have the potential to reduce the operating costs for upstream production substantially compared to cell cultures. In the transient expression approach, wild-type plants are vacuum infiltrated with Agrobacterium tumefaciens carrying a vector encoding the mAb heavy and light chains, allowing large quantities of mAb to accumulate within 5-8 days (Shoji et al., 2012). Although this method is rapid, its potential drawback for VLS production is that each plant in every batch must be infiltrated with bacteria, which merely transfers the fermentation costs to the production of bacteria (Houdelet et al., 2017). ...
Chapter
The very-large-scale (VLS) production of monoclonal antibodies (mAbs) in plants appears to have several advantages over cell culture platforms for upstream production, whereas the downstream capture and polishing steps would be similar to those already used for the purification of mAbs from Chinese hamster ovary (CHO) cells, with similar cost implications. However, extraction and clarification, the process steps between upstream production and purification, differ substantially between plant- and fermentation-based processes. This chapter outlines the existing diversity of plant-based manufacturing processes and explain why it is important to establish predictive and scalable models for each operation. It discusses the aspects of process diversity that can be consolidated to facilitate cost-effective scale-up. Finally, the chapter considers how the VLS plant-based production of mAbs may broaden the potential of our healthcare systems. The plant biotechnology community can learn and benefit from the expertise developed during the optimization of CHO cells, particularly with regard to chromatography.
Article
Nanoparticles are used as carriers for the delivery of drugs and imaging agents. Proteins are safer than synthetic nanocarriers due to their greater biocompatibility and absence of toxic degradation products. In this context, ferritin has the additional benefit of inherently targeting the membrane receptor transferrin 1, which is overexpressed by most cancer cells. Furthermore, this self-assembling multimeric protein can be loaded with more than 2000 iron atoms, as well as drugs, contrast agents and other cargos. However, recombinant ferritin currently costs ~3.5 million € g⁻¹, presumably because the limited number of producers cannot meet demand, making it generally unaffordable as a nanocarrier. Because plants can produce proteins at very-large-scale, we developed a simple, proof-of-concept process for the production of the human ferritin heavy chain (FTH1) by transient expression in Nicotiana benthamiana. We optimized the protein yields by screening different compartments and 5′-UTRs in plant cell packs, and selected the best-performing construct for production in differentiated plants. We then established a rapid and scalable purification protocol by combining pH and heat treatment before extraction, followed by a ultrafiltration/diafiltration size-based separation process. The optimized process achieved ferritin levels of ~40 mg kg⁻¹ fresh biomass although depth filtration limited product recovery to ~7%. The purity of the recombinant product was >90% at costs ~ 3% of the current sales price. Our method therefore allows the production of affordable ferritin heavy chain as a carrier for therapeutic and diagnostic agents, which is suitable for further stability and functionality testing in vitro and in vivo. This article is protected by copyright. All rights reserved.
Article
Immobilized metal affinity chromatography (IMAC) ensures the specific purification of proteins containing histidine tags through high affinity with transition metal chelators, which has various applications in biological protein separation. Most chromatographic separations currently use a fixed bed. In this form, internal flow pressure drops very sharply, accompanied by uneven solution flow, pore blockages, etc., all of which greatly reduce separation efficiency. Therefore, this study uses hollow fiber membranes (HFMs) with micron-scale inner diameters as a base, thus reducing operating pressure and significantly enhancing mass transmission. Batch adsorption experiments were performed using flat plate membranes to obtain the reaction's thermodynamic and kinetic model parameters for use in a dynamic column breakthrough simulation. The numerical simulation was based on a single HFM model and established a mathematical model for computational fluid dynamics (CFD) in ANSYS Fluent software. Model accuracy was validated by combining the simulation with experiments. The effects of different module and process parameters on the breakthrough curve were investigated by varying parameters such as flow rate, initial feed concentration, and HFM inner diameter. Design parameters and operating conditions contributing to module utilization were subsequently obtained.
Article
Plant-based production systems are inexpensive and easy to handle, allowing them to complement existing platforms for the production of protein-based vaccines, therapeutics and diagnostic reagents. However, screening product candidates in whole plants requires a large facility footprint and is challenging due to natural variations in recombinant protein accumulation. In contrast, plant cell packs (PCPs) allow more than 1000 samples to be screened per day in microtiter plates. PCPs enable rapid development cycles based on transient expression in as little as 3 days, and yield milligram quantities of product for initial quality assessment and functional testing. However, this requires high-level expression in BY-2 cells and consistent cell quality across batches. We therefore used a statistical design of experiments (DoE) approach to systematically assess factors that contribute to consistent high yields of recombinant proteins in PCPs. Specifically, we tested the osmolality, pH, carbon source, light source, and additives during cell cultivation, as well as cell and PCP harvest times. The careful adjustment of these factors increased overall productivity by approximately fourfold. Remarkably all cultivation conditions leading to high productivities during transient expression in PCPs were associated with a reduced water uptake into the central vacuole. The universal presence of a vacuole in plant cells indicates that our results should be transferrable to other cells lines. Our findings therefore support the broad application of PCPs for screening and product analysis during the development of protein-based pharmaceuticals and reagents in plants.
Article
The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid wild relative of tobacco used for transient protein expression in laboratory and commercial settings. To improve the performance of this species, we established a multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants. As proof of principle, we targeted the P4H4 gene encoding a prolyl‐4‐hydroxylase involved in protein O‐linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, we established transgenic plant lines with knockout mutations in all four P4H4 genes simultaneously. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and we subsequently identified transgene‐free lines with homozygous or biallelic mutations. The analysis of plant‐produced recombinant IgA1 as a reporter protein revealed changes in the amount of peptides containing hydroxyproline residues and pentoses in the knockout plants. The multiplex expression of efficient gRNAs combined with the DsRed2 marker in a binary vector system reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes needed to obtain transgene‐free progeny. This article is protected by copyright. All rights reserved
Chapter
Transient protein expression in plant cells is less time consuming than the production of whole transgenic plants. For transient expression, agroinfiltration is a simple and effective method to deliver transgenes into plant cells. After an Agrobacterium infection, recombinant proteins can be produced in plant cells from 3 to 10 days. To increase protein yield, a deconstructed viral vector has been used. This chapter provides a detailed description of the transient expression of recombinant proteins in a well-developed host strain of Nicotiana benthamiana. This study also describes the necessary steps for the extraction of soluble proteins from agroinfiltrated leaves.
Chapter
Plants have always played a predominant role in both the nutritional and medical components of human health. Innovations in plant biology, in particular plant biotechnology, have helped these disciplines advance significantly. While some of our modern drugs have their origins in plant species, many pharmaceuticals that are commercially available today using conventional production systems such as yeast or mammalian cells can now be generated within the plant material itself, through a novel approach known as molecular pharming. This chapter describes the use of plants as a production platform to generate medicines to address infectious and chronic diseases. Examples of biologics produced in plants, from vaccines, monoclonal antibodies to therapeutic agents are described. The development of nanoparticles for cancer therapy based on plant viruses is also discussed. The chapter concludes with a projection of future applications for plant-based pharmaceuticals.
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Plants are platforms where recombinant proteins and other biopharmaceuticals can be produced easily, cheaply and safely and can be purified back. Recently, many recombinant proteins such as growth hormone, antibody milk proteins, serum albumin and various industrial enzymes produced in bacterial or mammalian cell cultures have been produced in plant tissue or in plant cell culture. Plant tissues provide suitable post-translational modifications for production of recombinant viral and bacterial antigens and show the same biological activity as the recombinant vaccines produced in microorganisms. All of these have paved the way for their usage in vaccine production. Production of recombinant protein in plants requires stable or transient integration of target gene sequence depending on the location in the plant cell. While the biolistic method is used for the stable transformation of the target gene in the nucleus or chloroplast, plant pathogen Agrobacterium sp. mediated gene transfer method is used for transient gene transfer. Plant system are extremely suitable expression vectors for industrial production of pharmaceutical proteins, with their proven production capacity and economic feasibility. Keywords: plant derived vaccines, genetic modification, plant biotechnology--Bitkisel türevli aşılar Özet Bitkiler, rekombinant proteinlerin ve diğer biyofarmasotiklerin kolay, ucuz ve güvenli üretiminin sağlanabildiği ve geri saflaştırılabildiği platformlardır. Günümüzde bakteri ya da memeli hücre kültürlerinde üretilen büyüme hormonu, antikor süt proteinleri, serum albumini ve çeşitli endüstriyel enzimler gibi birçok rekombinant proteinin bitkisel dokuda ya da bitki hücre kültüründe üretimi gerçekleştirilmiştir. Bitkisel dokuların rekombinant viral ve bakteriyel antijenlerin üretimleri için uygun post-translasyonel modifikasyonları sağlamaları ve mikroorganizmalarda üretilen rekombinant aşılar ile aynı biyolojik aktiviteyi göstermeleri, aşı üretiminde kullanılmalarının önünü açmıştır. Bitkisel rekombinant protein üretimi, hedef gen dizisinin bitki hücresindeki konumuna bağlı olarak stabil veya geçici entegrasyonunu gerektirir. Biyolistik yöntemi, hedef genin çekirdek veya kloroplasta stabil transformasyonu için kullanılırken, bir bitki patojeni olan Agrobacterium sp. aracılı gen transferi yöntemi, geçici ve stabil gen transferi için kullanılmaktadır. Bitkisel sistemler kanıtlanmış üretim kapasiteleri ve ekonomik fizibiliteleri ile farmasötik proteinlerin endüstriyel boyutta üretimleri için son derece uygun ekspresyon vektörleridir.
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Plant proteins can be used for the production of a variety of bioproducts, including films and coatings, adhesives, fibres and pharmaceuticals. Proteins derived from plant production systems have many advantages: they are safe, low-cost and rapidly deployable, allow for simple product storage and result in proteins that are properly folded, assembled and post-translationally modified. While plant-derived protein-based products are natural, renewable, biodegradable and environmentally friendly, they tend to be lower in strength and elasticity than their corresponding synthetic products. Current research in this area is focused on overcoming challenges in plant production platforms related to yield, purification, regulatory approval and customer acceptance.
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The yield of recombinant proteins in plants determines their economic competitiveness as a production platform compared to microbes and mammalian cells. The promoter, untranslated regions (UTRs) and codon usage can all contribute to the yield, but potential interactions among these components have not been examined in detail. Here we investigated the effect of two promoters (35SS and nos) and four 5′UTRs on the spatiotemporal expression of DsRed mRNA and the accumulation of DsRed protein during transient expression in tobacco (Nicotiana tabacum) mediated by Agrobacterium tumefaciens. We found that the mRNA levels peaked 2‐3 days post‐infiltration (dpi), and rapidly declined thereafter, whereas DsRed protein was first detected after ∼3 days and concentrations continued to increase until at least 5 dpi. This temporal decoupling of mRNA and protein expression was strongest in the older leaves, which also produced the lowest DsRed yields. The accumulation of DsRed linearly correlated with mRNA levels in all but the youngest leaves, where more DsRed was synthesized per mRNA molecule. This was the case for both promoters, although the nos promoter had a higher protein/mRNA ratio than the 35SS promoter. Furthermore, the type of 5′UTR affected DsRed protein accumulation by 50% starting from similar levels of mRNA. We concluded that DsRed mRNA levels are not the limiting factor for DsRed protein expression in plants, but that translation‐associated processes such as initiation, elongation and release are bottlenecks that should be addressed in future studies.
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Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.
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Herbal remedies were the first medicines used by humans due to the many pharmacologically active secondary metabolites produced by plants. Some of these metabolites inhibit cell division and can therefore be used for the treatment of cancer, e.g. the mitostatic drug paclitaxel (Taxol). The ability of plants to produce medicines targeting cancer has expanded due to the advent of genetic engineering, particularly in recent years because of the development of gene editing systems such as the CRISPR/Cas9 platform. These technologies allow the introduction of genetic modifications that facilitate the accumulation of native pharmaceutically-active substances, and even the production heterologous recombinant proteins, including human antibodies, lectins and vaccine candidates. Here we discuss the anti-cancer agents that are produced by plants naturally or following genetic modification, and the potential of these products to supply modern healthcare systems. Special emphasis will be put on proteinaceous anti-cancer agents, which can exhibit an improved selectivity and reduced side effects compared to small molecule-based drugs.
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Every year the growth of the poultry industry is severely threatened by a number of infectious viral, bacterial and parasitic diseases. There are a number of vaccines to control these diseases including inactivated virus vaccines, attenuated virus vaccines, live virus vaccines, and subunit vaccines, but they are often relatively expensive and require cold storage and trained people to administer them, especially in developing countries. Plant-based vaccines provide a better option to control these diseases in low profit margin poultry industry. Still there are some challenges in the field of plant-based, so called ‘green’ vaccines. Injection-based oral priming is a big challenge for commercialisation of green vaccines so, new techniques are needed in the field of plant-based vaccine to pass these barriers for commercialisation. This discusses the potential for plant-based vaccines and whether they are good option to control poultry diseases.
Chapter
The use of plants as efficient biopharmaceutical factories has significantly increased in the past two decades. This is mainly due to advancements in plant biotechnology which pave the way to high-yield production of biopharmaceuticals in plants, combined with efforts made to optimize yield through upstream, downstream, and preservation strategies of recombinant proteins. The FDA’s approval to commercially release recombinant glucocerebrosidase enzyme produced in carrot cells by Protalix Biotherapeutics was the first plant-produced biopharmaceutical to be released for human consumption into the market. This is a major achievement in the field of molecular pharming. Although many other biopharmaceuticals produced in plants are in the pipeline for commercial release after undergoing various stages of clinical trials, there is room for improvement in enhancing recombinant protein yield in plants. These include exploration of innovative strategies involving genetics, genomics, epigenetics, in silico simulations and purification techniques. In this chapter, we discuss various approaches of plant biotechnology and plant genetic engineering that are being used in the molecular pharming of biopharmaceuticals.
Chapter
Plants were the first sources of medicines used by humankind, with evidence of herbal remedies dating back at least 60,000 years. Many plants have been used medicinally because they produce secondary metabolites with pharmacological properties, including compounds such as paclitaxel (Taxol) that inhibit cell division and can therefore be used as a treatment for cancer. With the advent of recombinant DNA and molecular biotechnology in the 1970s, plants have also been modified genetically to produce more of their native pharmaceutically active substances, or even nonnative compounds. The scope of medicinal plants has also expanded beyond secondary metabolites to include pharmaceutical recombinant proteins, such as human antibodies. This chapter provides an overview of the anticancer compounds naturally produced in plants and how gene technology has been used to facilitate their production. It also considers how plant-based expression systems can help to supply modern healthcare systems with protein-based anticancer compounds such as monoclonal antibodies, lectins, and anticancer vaccines.
Chapter
The use of plants as efficient biopharmaceutical factories has significantly increased in the past two decades. This is mainly due to advancements in plant biotechnology which pave the way to high-yield production of biopharmaceuticals in plants, combined with efforts made to optimize yield through upstream, downstream, and preservation strategies of recombinant proteins. The FDA’s approval to commercially release recombinant glucocerebrosidase enzyme produced in carrot cells by Protalix Biotherapeutics was the first plant-produced biopharmaceutical to be released for human consumption into the market. This is a major achievement in the field of molecular pharming. Although many other biopharmaceuticals produced in plants are in the pipeline for commercial release after undergoing various stages of clinical trials, there is room for improvement in enhancing recombinant protein yield in plants. These include exploration of innovative strategies involving genetics, genomics, epigenetics, in silico simulations and purification techniques. In this chapter, we discuss various approaches of plant biotechnology and plant genetic engineering that are being used in the molecular pharming of biopharmaceuticals.
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In 2009, a novel H1N1 swine influenza virus was isolated from infected humans in Mexico and the United States, and rapidly spread around the world. Another virus, a highly pathogenic avian influenza virus of the H5N1 subtype, identified by the World Health Organization as a potential pandemic threat in 1997, continues to be a significant risk. While vaccination is the preferred strategy for the prevention and control of influenza infections, the traditional egg-based approach to producing influenza vaccines does not provide sufficient capacity and adequate speed to satisfy global needs to combat newly emerging strains, seasonal or potentially pandemic. Significant efforts are underway to develop and implement new cell substrates with improved efficiency for influenza vaccine development and manufacturing. In recent years, plants have been used to produce recombinant proteins including subunit vaccines and antibodies. The main advantages of using plant systems for the production of vaccine antigens against influenza are their independence from pathogenic viruses, and cost and time efficiency. Here, we describe the large-scale production of recombinant hemagglutinin proteins from A/California/04/09 (H1N1) and A/Indonesia/05/05 (H5N1) strains of influenza virus in Nicotiana benthamiana plants, and their immunogenicity (serum hemagglutination inhibition and virus neutralizing antibodies), and safety in animal models. These results support the testing of these candidate vaccines in human volunteers and also the utility of our plant expression system for large-scale recombinant influenza vaccine production.
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The reappearance of highly pathogenic avian influenza H5N1 in poultry in 2003, and the subsequent high-fatality zoonoses in Asia, Europe and Africa, has heightened the awareness of a potential pandemic and the need for global vaccine supply. Most manufacturers still use embryonated hens' eggs to produce influenza vaccines, a system that has demonstrated its value throughout six decades. There are, however, some challenges with this approach, both for seasonal and particularly for pandemic vaccine production. This review highlights some of these challenges and describes emerging alternative production platforms with the potential to deliver safe and effective vaccines to the global market in a timely fashion. A particular emphasis of this review will be on the production of recombinant influenza vaccines using transient plant expression systems.
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The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.
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The global spread of highly pathogenic avian influenza virus (H5N1 subtype) has promoted efforts to develop human vaccines against potential pandemic outbreaks. However, current platforms for influenza vaccine production are cumbersome, limited in scalability and often require the handling of live infectious virus. We describe the production of hemagglutinin from the A/Indonesia/05/05 strain of H5N1 influenza virus by transient expression in plants, and demonstrate the immunogenicity and protective efficacy of the vaccine candidate in animal models. Immunization of mice and ferrets with plant-derived hemagglutinin elicited serum hemagglutinin-inhibiting antibodies and protected the ferrets against challenge infection with a homologous virus. This demonstrates that plant-derived H5 HA is immunogenic in mice and ferrets, and can induce protective immunity against infection with highly pathogenic avian influenza virus. Plants could therefore be suitable as a platform for the rapid, large-scale production of influenza vaccines in the face of a pandemic.
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Cell substrates are a key component of successful vaccine development and throughout the last several decades there has been a dramatic increase in the types of cells available for vaccine production. Nevertheless, there is a continued demand for new and innovative approaches for vaccine development and manufacturing. Recent developments involving cells of insect and plant origin are attracting considerable scientific interest. Here we review vaccine antigen production in plant-based systems as was presented by Dr. Vidadi Yusibov of Fraunhofer USA Center for Molecular Biotechnology at the IABS International Scientific Workshop on NEW CELLS FOR NEW VACCINES II that was held in Wilmington, Delaware on September 17-19, 2007.
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Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.
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The H3N2 influenza A/Fujian/411/02-like virus strains that circulated during the 2003-2004 influenza season caused influenza epidemics. Most of the A/Fujian/411/02 virus lineages did not replicate well in embryonated chicken eggs and had to be isolated originally by cell culture. The molecular basis for the poor replication of A/Fujian/411/02 virus was examined in this study by the reverse genetics technology. Two antigenically related strains that replicated well in embryonated chicken eggs, A/Sendai-H/F4962/02 and A/Wyoming/03/03, were compared with the prototype A/Fujian/411/02 virus. A/Sendai differed from A/Fujian by three amino acids in the neuraminidase (NA), whereas A/Wyoming differed from A/Fujian by five amino acids in the hemagglutinin (HA). The HA and NA segments of these three viruses were reassorted with cold-adapted A/Ann Arbor/6/60, the master donor virus for the live attenuated type A influenza vaccines (FluMist). The HA and NA residues differed between these three H3N2 viruses evaluated for their impact on virus replication in MDCK cells and in embryonated chicken eggs. It was determined that replication of A/Fujian/411/02 in eggs could be improved by either changing minimum of two HA residues (G186V and V226I) to increase the HA receptor-binding ability or by changing a minimum of two NA residues (E119Q and Q136K) to lower the NA enzymatic activity. Alternatively, recombinant A/Fujian/411/02 virus could be adapted to grow in eggs by two amino acid substitutions in the HA molecule (H183L and V226A), which also resulted in the increased HA receptor-binding activity. Thus, the balance between the HA and NA activities is critical for influenza virus replication in a different host system. The HA or NA changes that increased A/Fujian/411/02 virus replication in embryonated chicken eggs were found to have no significant impact on antigenicity of these recombinant viruses. This study demonstrated that the reverse genetics technology could be used to improve the manufacture of the influenza vaccines.
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Knowledge about the complete genome constellation of seasonal influenza A viruses from different countries is valuable for monitoring and understanding of the evolution and migration of strains. Few complete genome sequences of influenza A viruses from Europe are publicly available at the present time and there have been few longitudinal genome studies of human influenza A viruses. We have studied the evolution of circulating human H3N2, H1N1 and H1N2 influenza A viruses from 1999 to 2006, we analysed 234 Danish human influenza A viruses and characterised 24 complete genomes. H3N2 was the prevalent strain in Denmark during the study period, but H1N1 dominated the 2000-2001 season. H1N2 viruses were first observed in Denmark in 2002-2003. After years of little genetic change in the H1N1 viruses the 2005-2006 season presented H1N1 of greater variability than before. This indicates that H1N1 viruses are evolving and that H1N1 soon is likely to be the prevalent strain again. Generally, the influenza A haemagglutinin (HA) of H3N2 viruses formed seasonal phylogenetic clusters. Different lineages co-circulating within the same season were also observed. The evolution has been stochastic, influenced by small "jumps" in genetic distance rather than constant drift, especially with the introduction of the Fujian-like viruses in 2002-2003. Also evolutionary stasis-periods were observed which might indicate well fit viruses. The evolution of H3N2 viruses have also been influenced by gene reassortments between lineages from different seasons. None of the influenza genes were influenced by strong positive selection pressure. The antigenic site B in H3N2 HA was the preferred site for genetic change during the study period probably because the site A has been masked by glycosylations. Substitutions at CTL-epitopes in the genes coding for the neuraminidase (NA), polymerase acidic protein (PA), matrix protein 1 (M1), non-structural protein 1 (NS1) and especially the nucleoprotein (NP) were observed. The N-linked glycosylation pattern varied during the study period and the H3N2 isolates from 2004 to 2006 were highly glycosylated with ten predicted sequons in HA, the highest amount of glycosylations observed in this study period. The present study is the first to our knowledge to characterise the evolution of complete genomes of influenza A H3N2, H1N1 and H1N2 isolates from Europe over a time period of seven years from 1999 to 2006. More precise knowledge about the circulating strains may have implications for predicting the following season strains and thereby better matching the vaccine composition.
Article
Historically, most vaccines have been based on killed or live-attenuated infectious agents. Although very successful at immunizing populations against disease, both approaches raise safety concerns and often have limited production capacity. This has resulted in increased emphasis on the development of subunit vaccines. Several recombinant systems have been considered for subunit vaccine manufacture, including plants, which offer advantages both in cost and in scale of production. We have developed a plant expression system utilizing a 'launch vector', which combines the advantageous features of standard agrobacterial binary plasmids and plant viral vectors, to achieve high-level target antigen expression in plants. As an additional feature, to aid in target expression, stability and purification, we have engineered a thermostable carrier molecule to which antigens are fused. We have applied this launch vector/carrier system to engineer and express target antigens from various pathogens, including, influenza A/Vietnam/04 (H5N1) virus.
Article
Plant-produced vaccines are a much-hyped development of the past two decades, whose time to embrace reality may have finally come. Vaccines have been developed against viral, bacterial, parasite and allergenic antigens, for humans and for animals; a wide variety of plants have been used for stable transgenic expression as well as for transient expression via Agrobacterium tumefaciens and plant viral vectors. A great many products have shown significant immunogenicity; several have shown efficacy in target animals or in animal models. The realised potential of plant-produced vaccines is discussed, together with future prospects for production and registration.
Article
Seasonal influenza continues to have a huge annual impact in the United States, accounting for tens of millions of illnesses, hundreds of thousands of excess hospitalizations, and tens of thousands of excess deaths. Vaccination remains the mainstay for the prevention of influenza. In the United States, 2 types of influenza vaccine are currently licensed: trivalent inactivated influenza vaccine and live attenuated influenza vaccine. Both are safe and effective in the populations for which they are approved for use. Children, adults <65 years of age, and the elderly all receive substantial health benefits from vaccination. In addition, vaccination appears to be cost-effective, if not cost saving, across the age spectrum. Despite long-standing recommendations for the routine vaccination of persons in high-priority groups, US vaccination rates remain too low across all age groups. Important issues to be addressed include improving vaccine delivery to current and expanded target groups, ensuring timely availability of adequate vaccine supply, and development of even more effective vaccines. Development of a vaccine against potentially pandemic strains is an essential part of the strategy to control and prevent a pandemic outbreak. The use of existing technologies for influenza vaccine production would be the most straightforward approach, because these technologies are commercially available and licensing would be relatively simple. Approaches currently being tested include subvirion inactivated vaccines and cold-adapted, live attenuated vaccines. Preliminary results have suggested that, for some pandemic antigens, particularly H5, subvirion inactivated vaccines are poorly immunogenic, for reasons that are not clear. Data from evaluation of live pandemic vaccines are pending. Second-generation approaches designed to provide improved immune responses at lower doses have focused on adjuvants such as alum and MF59, which are currently licensed for influenza or other vaccines. Additional experimental approaches are required to achieve the ultimate goal for seasonal and pandemic influenza prevention--namely, the ability to generate broadly cross-reactive and durable protection in humans.
Article
Variability in growth characteristics of influenza B viruses remains a serious limitation in the manufacture of inactivated influenza vaccines. Currently, serial passage in eggs is the strategy used in most instances for selection of high growth virus variants. In previous studies we found that adaptation of the strain B/Victoria/504/2000 to high growth in eggs was associated with changes only in hemagglutinin (HA). The high growth phenotype was associated with acquisition of either two (R162M and D196Y) or three (G141E, R162M and D196Y) amino acid (AA) substitutions, predicted to be near the receptor-binding domain of HA. In the present study we analyzed, using reverse genetics, the contribution to virus growth of each of these AA substitutions and determined their effect on antigenic properties. We found that G141E and R162M were most favorable for virus growth; however, only R162M could improve virus growth without antigenic alteration. Substitution D196Y had least effect on virus growth but substantially altered antigenic properties. Additional virus variants with AA substitutions at positions 126, 129, 137 and 141 were generated and characterized. The AA changes advantageous for growth of B/Victoria/504/2000 were also tested in the context of the HA of the B/Beijing/184/93, a virus with stable low-growth phenotype. All of the tested AA substitutions improved the replicative capabilities of the corresponding viruses, but only N126D and K129E had no effect on antigenicity. The results of our studies demonstrate that introduction of specific AA substitutions into viral HA can improve viral replicative efficiency while preserving the original antigenic properties.
Article
Subunit vaccine production is typically associated with bacterial, yeast, insect or mammalian cell culture systems. Plants, however, are emerging as an alternative platform for producing vaccine antigens, and offer some advantages over other recombinant systems. In particular, plant virus-based transient expression systems are suitable for rapid engineering, ease of scale-up and cost-effective production of target antigens. In addition, this system provides an ideal approach for producing large quantities of vaccine antigens in a short period of time, which is particularly important when faced with natural outbreaks or accidental or intended release of bio-threat agents such as Bacillus anthrax and Yersinia pestis. This commentary reviews the production and evaluation of antigens made in plants in an attempt to develop vaccines against B. anthracis and Y. pestis.
Article
Influenza is a globally important respiratory pathogen that causes a high degree of morbidity and mortality annually. Although current vaccines are effective against virus infection, new strategies need to be developed to satisfy the global demand for an influenza vaccine. To address this point, we have engineered and produced the full-length hemagglutinin (HA) protein from the A/Wyoming/03/03 (H3N2) strain of influenza in plants. The antigenicity of this plant-produced HA was confirmed by ELISA and single-radial immunodiffusion (SRID) assays. Immunization of mice with plant-produced HA resulted in HA-specific humoral (IgG1, IgG2a and IgG2b) and cellular (IFNgamma and IL-5) immune responses. In addition, significant serum hemagglutination inhibition (HI) and virus neutralizing (VN) antibody titers were obtained with an antigen dose as low as 5mug. These results demonstrate that plant-produced HA protein is antigenic and can induce immune responses in mice that correlate with protection.
Plant-produced vaccines: promise and reality
  • E P Rybicki
Rybicki EP. Plant-produced vaccines: promise and reality. Drug Discov Today 2009; 14:16-24.
Fraunhofer USA CMB Plant Produced H1N1 Vaccine Begins Phase 1 Clinical Trial
  • Usa Fraunhofer
  • Center For Molecular
  • Biotechnology
Fraunhofer USA Center for Molecular Biotechnology. Fraunhofer USA CMB Plant Produced H1N1 Vaccine Begins Phase 1 Clinical Trial. Available at http://www.businesswire.com/news/home/ 20100917006118/en/Fraunhofer-USA-CMB-Plant-Produced-H1N1-Vaccine (Accessed 17 September 2010).
Transient protein expression in three Pisum sativum (green pea) varieties
  • Bj Green
  • M Fujiki
  • V Mett
  • Etal
Plant-based rapid production of recombinant subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza
  • Y Shoji
  • Ja Chichester
  • M Jones
  • Etal
Plant Produced H1N1 Vaccine Begins Phase 1 Clinical Trial http
  • Usa Fraunhofer
  • Usa Center For Molecular Biotechnology Fraunhofer
  • Cmb
Chichester and V. Mett wrote the paper
  • Y Shoji
  • C E Farrance
Y. Shoji, C. E. Farrance, J. A. Chichester and V. Mett wrote the paper.
Serum cross-reactive antibody response to a novel influenza A (H1N1) virus after vaccination with seasonal influenza vaccine
  • Centers for Disease Control and Prevention