Potential of plants to produce recombinant protein products

Plants have great potential as photosynthetic factories to produce pharmaceutically important and commercially valuable biomedicines and industrial proteins at low cost. The U.S. Food and Drug Administration (U.S. FDA) has approved the drug Elelyso (taliglucerase alfa) produced by carrot cells for treatment of type 1 Gaucher’s disease in 2012. The commercial potential of biomedicines produced by molecular farming has dramatically improved due to the success of an experimental drug called ZMapp, which has immunological activity in Ebola patients. A cocktail of three monoclonal antibodies was produced in tobacco (Nicotiana benthamiana) plants (Chen and Davis 2016). At present, very few drugs made by this technology have been approved by worldwide authorities such as the U.S. FDA. However, plants have been proposed as a novel paradigm for commercial production of proteins over the next decade. In recent years, leading researchers on molecular farming have given more priority to the area of animal-free therapeutic proteins such as parenteral and oral vaccines. Although plant-based platforms have considerable advantages over traditional systems such as bacterial and animal systems, there are several obstacles to commercial-scale production, especially with regards to improving the quality and quantity of plant-produced biologics and industrial materials. One of the biggest barriers to commercialization of this technology is the intense scrutiny of these new plant varieties by regulatory agencies and the public as well as the high costs associated with their regulatory approval.     

Benefits and risks of antibody and vaccine production in transgenic plants

Phytopharming, the production of protein biologicals in recombinant plant systems, has shown great promise in studies performed over the past 13 years. A secretory antibody purified from transgenic tobacco was tested successfully in humans, and prevented bacterial re-colonization after topical application in the mouth. Rapid production of patient-tailored anti-lymphoma antibodies in recombinant Tobamovirus-infected tobacco may provide effective cancer therapy. Many different candidate vaccines from bacterial and viral sources have been expressed in transgenic plants, and three human clinical trials with oral delivery of transgenic plant tissues have shown exciting results. The use of crop plants with agricultural practice could allow cheap production of valuable proteins, while providing enhanced safety by avoidance of animal viruses or other contaminants. However development of this technology must carefully consider the means to ensure the separation of food and medicinal products when crop plants are used for phytopharming.     

植物口服疫苗的研究进展

植物口服疫苗是通过转基因植物生产,通过口服的方式预防疾病的生物制品。作为一种新型疫苗,其研究开始于三十几年前。由于植物口服疫苗可以最大程度地降低传统疫苗的潜在风险,在疫苗生产中具有优势,因此拥有良好的商业生产前景。植物疫苗价格低廉,生产过程安全,可产生与注射疫苗相似效价效果,无论是在控制养殖业抗生素滥用的情况下作为替代方法,还是在某些经济发展水平不高、卫生条件较差的发展中国家用于预防和控制某些传染病,都是十分理想的。该文对植物口服疫苗生产方法、候选生物反应器、疫苗有效性、适用范围和发展前景进行了概述。此外,还着重对目前开展的植物口服疫苗在病毒、细菌、寄生虫引起的人畜共患病中的应用研究,以及其在人类肿瘤预防中所开展的应用研究进行了较为详细的综述。虽然植物疫苗的研究及应用在植物外源基因表达量、免疫剂量、免疫接种途径等方面还存在很多挑战,但依然为传统疫苗学的研发提供了一条充满希望的新途径。     

Transgenic plants for animal health: plant-made vaccine antigens for animal infectious disease control

A variety of plant species have been genetically modified to accumulate vaccine antigens for human and animal health and the first vaccine candidates are approaching the market. The regulatory burden for animal vaccines is less than that for human use and this has attracted the attention of researchers and companies, and investment in plant-made vaccines for animal infectious disease control is increasing. The dosage cost of vaccines for animal infectious diseases must be kept to a minimum, especially for non-lethal diseases that diminish animal welfare and growth, so efficient and economic production, storage and delivery are critical for commercialization. It has become clear that transgenic plants are an economic and efficient alternative to fermentation for large-scale production of vaccine antigens. The oral delivery of plant-made vaccines is particularly attractive since the expensive purification step can be avoided further reducing the cost per dose. This review covers the current status of plant-produced vaccines for the prevention of disease in animals and focuses on barriers to the development of such products and methods to overcome them.     

Overview of marker vaccine and differential diagnostic test technology.

Recent advances in molecular biology, immunology, microbiology, genetics and microbial pathogenesis have lead to the development of a wide variety of new approaches for developing safer and more effective vaccines based on designs such as subunit vaccines, gene deleted vaccines, live vectored vaccines, and DNA mediated vaccines. Technology tools can be as basic as identifying naturally occurring strains with deletions that support differentiating infected from vaccinated animal (DIVA) needs or be based on higher technology developments such as improved protein expression and purification methods, transgenic plant- and plant virus-based antigen production, and novel adjuvants that target specific immune responses. These new approaches, when applied to the development of marker vaccines and companion diagnostic test kits hold tremendous potential for developing improved tools for eradication and control programs. Marker vaccines and companion diagnostic test kits must meet the established licensing requirements for purity, potency, safety and efficacy. Efficacy claims are based on evaluation of the level of protection demonstrated in host animal trials and may range from "prevents infection with (a specific agent)", to "for use as an aid in the reduction of disease due to (a specific agent)." The differences in claims and recommendations are a function of the variation in protection elicited by various vaccines. For designing effective eradication programs, vaccine efficacy characteristics such as for reducing susceptibility to infections and spread of infections must be well defined; similarly, diagnostic test performance characteristics (efficacy) must be determined. In addition to data to support efficacy claims, it is imperative that safety of production and use of vaccines be evaluated. During the design of marker vaccines and diagnostic tests, it is important to consider the application of appropriate technologies to improve the safety of these products. Use of recombinant technologies for production of vaccines and/or diagnostic test antigens can reduce the biosafety concerns during production and during use, including human exposure to zoonotic pathogens during production and use, and potential spread of foreign animal disease agents due to loss of biocontainment. In addition, vaccines may induce adverse reactions. It is important to determine the frequency of adverse events and to reduce the likelihood of induction of adverse reactions through proper design.     

Selectable marker genes in transgenic plants: applications, alternatives and biosafety

Approximately fifty marker genes used for transgenic and transplastomic plant research or crop development have been assessed for efficiency, biosafety, scientific applications and commercialization. Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional on the presence of external substrates. Positive selectable marker genes are defined as those that promote the growth of transformed tissue whereas negative selectable marker genes result in the death of the transformed tissue. The positive selectable marker genes that are conditional on the use of toxic agents, such as antibiotics, herbicides or drugs were the first to be developed and exploited. More recent developments include positive selectable marker genes that are conditional on non-toxic agents that may be substrates for growth or that induce growth and differentiation of the transformed tissues. Newer strategies include positive selectable marker genes which are not conditional on external substrates but which alter the physiological processes that govern plant development. A valuable companion to the selectable marker genes are the reporter genes, which do not provide a cell with a selective advantage, but which can be used to monitor transgenic events and manually separate transgenic material from non-transformed material. They fall into two categories depending on whether they are conditional or non-conditional on the presence of external substrates. Some reporter genes can be adapted to function as selectable marker genes through the development of novel substrates. Despite the large number of marker genes that exist for plants, only a few marker genes are used for most plant research and crop development. As the production of transgenic plants is labor intensive, expensive and difficult for most species, practical issues govern the choice of selectable marker genes that are used. Many of the genes have specific limitations or have not been sufficiently tested to merit their widespread use. For research, a variety of selection systems are essential as no single selectable marker gene was found to be sufficient for all circumstances. Although, no adverse biosafety effects have been reported for the marker genes that have been adopted for widespread use, biosafety concerns should help direct which markers will be chosen for future crop development. Common sense dictates that marker genes conferring resistance to significant therapeutic antibiotics should not be used. An area of research that is growing rapidly but is still in its infancy is the development of strategies for eliminating selectable marker genes to generate marker-free plants. Among the several technologies described, two have emerged with significant potential. The simplest is the co-transformation of genes of interest with selectable marker genes followed by the segregation of the separate genes through conventional genetics. The more complicated strategy is the use of site-specific recombinases, under the control of inducible promoters, to excise the marker genes and excision machinery from the transgenic plant after selection has been achieved. In this review each of the genes and processes will be examined to assess the alternatives that exist for producing transgenic plants.     

可视安全标记花青素合成途径相关基因在植物转化研究中的应用进展

近年来有关转基因产品可能带来的环境和食品安全问题的争论多集中在作为标记基因的抗生素抗性基因以及抗除草剂基因的广泛使用。因此寻找安全、有效的标记基因替代上述有争议标记基因就显得十分必要和紧迫。花青素合成酶类及其合成调控因子可以控制植物体内的色素合成,一些转化研究已证明其转化体表型发生了颜色改变。加之花青素类物质是一些天然色素,对人类有利而无害,可以利用花青素的这些特点,将花青素合成酶类及其调控因子基因作为一类可视化、安全和有效的标记基因来转化植物。本文就可视安全标记花青素合成酶类基因及其调控基因在植物转化研究中的应用进展进行了简要介绍,以期为提高标记基因的安全性提供参考。     

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.     

Expression systems and developments in plant-made vaccines

Delivery of vaccines to mucosal surfaces can elicit humoral and cell-mediated responses of the mucosal and systemic immune systems, evoke less pain and discomfort than parenteral delivery, and eliminate needle-associated risks. Transgenic plants are an ideal means by which to produce oral vaccines, as the rigid walls of the plant cell protect antigenic proteins from the acidic environment of the stomach, enabling intact antigen to reach the gut associated lymphoid tissue. In the past few years, new techniques (such as chloroplast transformation and food processing) have improved antigen concentration in transgenic plants. In addition, adjuvants and targeting proteins have increased the immunogenicity of mucosally administered plant-made vaccines. These studies have moved plant-made vaccines closer to the development phase.     

Advances in selectable marker genes for plant transformation

Plant transformation systems for creating transgenics require separate process for introducing cloned DNA into living plant cells. Identification or selection of those cells that have integrated DNA into appropriate plant genome is a vital step to regenerate fully developed plants from the transformed cells. Selectable marker genes are pivotal for the development of plant transformation technologies because marker genes allow researchers to identify or isolate the cells that are expressing the cloned DNA, to monitor and select the transformed progeny. As only a very small portion of cells are transformed in most experiments, the chances of recovering transgenic lines without selection are usually low. Since the selectable marker gene is expected to function in a range of cell types it is usually constructed as a chimeric gene using regulatory sequences that ensure constitutive expression throughout the plant. Advent of recombinant DNA technology and progress in plant molecular biology had led to a desire to introduce several genes into single transgenic plant line, necessitating the development of various types of selectable markers. This review article describes the developments made in the recent past on plant transformation systems using different selection methods adding a note on their importance as marker genes in transgenic crop plants.     

Regulatory considerations for marker vaccines and diagnostic tests in the U.S.

Marker vaccines and diagnostic tests can prove to be invaluable in disease eradication and control programs, as was found in the pseudorabies (Aujeszky's Disease) virus eradication program in the U.S. During that campaign, numerous gene-deleted vaccines and companion diagnostic test kits were used to differentiate infected animals from vaccinated animals, in a strategy that ultimately led to eradication of the disease in commercial swine herds. The United States Department of Agriculture played a key role in delivery of that success by developing biologics policy, evaluating each product, and ensuring that the conditions of licensure were met. What was most critical in the overall eradication effort, however, was the detailed and dedicated interaction among key players: the biologics regulators, manufacturers, Federal, State, and local regulatory partners, veterinary researchers, industry associations, and animal owners. A good disease control program has to include all of these. The regulatory requirements for licensure of marker vaccines and diagnostic test kits are not different from that for other products. There are several mechanisms for vaccine approval, some more rapid than others, but only a few that could apply to these products. Generally, the platforms that might support marker vaccines and companion diagnostic kits are those based on genetic engineering or protein manipulation. If the product is derived from the application of biotechnology, then additional regulatory considerations are applicable. Most important of these are the considerations found in the National Environmental Policy Act (NEPA), wherein deliberate release of any organism containing recombinant DNA into the environment is subject to review and approval by appropriate federal agencies. Environmental release and NEPA compliance are discussed.     

Oral hepatitis B vaccine candidates produced and delivered in plant material

Hepatitis B is a major global health problem; approximately two billion people are infected with the virus worldwide, despite the fact that safe and efficacious vaccines have been developed and used for nearly 20 years. Prohibitive costs for vaccine purchase and administration restrict uptake in many developing nations. Agencies such as the Global Alliance for Vaccination and Immunization are helping to make current vaccines more available, but reduced costs would greatly aid this effort. Oral delivery is an option to reduce the expense of administering hepatitis B vaccines. It may also improve compliance, and orally delivered vaccines may be more efficacious among poor responders to current vaccines. However, to induce protective efficacy, oral administration may require encapsulation of antigen and delivery of large doses. Plant-based expression systems offer an oral delivery alternative with low production costs, and they also encapsulate the antigen. Some plant-based systems also stabilize antigen and therefore reduce storage and distribution costs. The hepatitis B major surface antigen has been expressed in several plant systems. A variety of regulatory sequences and subcellular targets have been used to achieve expression suitable for early stage clinical trials. However, further increase in expression will be necessary for practical and efficacious products. Appropriate processing can yield palatable products with uniform antigen concentration. The antigen expressed in plant systems shows extensive disulphide cross-linking and oligomerization and forms virus-like particles. Oral delivery of the antigen in plant material can induce a serum antibody response, prime the immune system for a subsequent injection of antigen and give a boosted response to a prior injection. Small scale clinical trials in which the antigen has been delivered orally in edible plant material indicate safety and immunogenicity.     

History of safe exposure and bioinformatic assessment of phosphomannose-isomerase (PMI) for allergenic risk

Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.

     

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.     

Heterologous protein production and delivery systems for Lactococcus lactis

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.     

Improved uptake of plant-derived LTB-linked proteins in carp gut and induction of specific humoral immune responses upon infeed delivery

Oral vaccination of fish is an effortless and stress free immunisation method which can be used for almost any age. However, vaccination via the mucosal route does have disadvantages. For example, the vaccine may induce tolerance and has to be protected to escape digestion. Also the vaccine should be efficiently delivered to immune-competent cells in the gut or other lymphoid organs. In addition, it should be cost effective. Here we present a novel fish vaccination model using potato tubers as vaccine production and delivery system. The model vaccines discussed here include fusion proteins consisting of a gut adhesion molecule (LTB) and a viral peptide or green fluorescent protein (GFP) expressed in potato tubers. The adhesion molecule mediates binding to and uptake from the gut, whereas the viral peptide or GFP functions as model vaccine antigen provoking the induction of an immune response. We demonstrate that fusion to LTB facilitates an elevated uptake of the model vaccines in carp gut mucosa. The plant-derived fusion proteins also elicit a specific systemic humoral immune response upon oral application of crude tuber material incorporated into a standard dietary feed pellet. The data presented here show the promising potentials of the plant as a production system for oral vaccines in aquaculture and feed mediated immunisation of fish.     

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.