High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

Background  

In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.     

High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts

Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55^gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55^gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55^gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55^gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55^gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. IGV publication no. 330     

Edible plant vaccines: applications for prophylactic and therapeutic molecular medicine

The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems. Genes encoding bacterial and viral antigens are faithfully expressed in edible tissues to form immunogenic proteins. Studies in animals and humans have shown that ingestion of transgenic plants containing vaccine proteins causes production of antigen-specific antibodies in serum and mucosal secretions. In general, the technology is limited by low expression levels for nuclear-integrated transgenes, but recent progress in plant organelle transformation shows promise for enhanced expression. The stability and immunogenicity of orally delivered antigens vary greatly, which necessitates further study on protein engineering to enhance mucosal delivery. These issues are discussed with regard to the further development of plant-based vaccine technology.     

Efficient and Stable Transformation of Lactuca sativa L. cv. Cisco (lettuce) Plastids

Transgenic plastids offer unique advantages in plant biotechnology, including high-level foreign protein expression. However, broad application of plastid genome engineering in biotechnology has been largely hampered by the lack of plastid transformation systems for major crops. Here we describe the development of a plastid transformation system for lettuce, Lactuca sativa L. cv. Cisco. The transforming DNA carries a spectinomycin-resistance gene (aadA) under the control of lettuce chloroplast regulatory expression elements, flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. On average, we obtained 1 transplastomic lettuce plant per bombardment. We show that lettuce leaf chloroplasts can express transgene-encoded GFP to ~36% of the total soluble protein. All transplastomic T0 plants were fertile and the T1 progeny uniformly showed stability of the transgene in the chloroplast genome. This system will open up new possibilities for the efficient production of edible vaccines, pharmaceuticals, and antibodies in plants.     

Expression of the rabies virus nucleoprotein in plants at high-levels and evaluation of immune responses in mice

Transgenic plants have been employed successfully as a low-cost system for the production of therapeutically valuable proteins including antibodies, antigens and hormones. Here, we report expression of a full-length nucleoprotein gene of rabies virus in transgenic tomato plants. The nucleoprotein was also transiently expressed in Nicotiana benthamiana plants by agroinfiltration. In both cases, the nucleoprotein was expressed at high levels, 1–5% of total soluble protein in tomato and 45% in N. benthamiana. Previously, only epitopes of the nucleoprotein had been expressed in plants. The presence and expression of the transgene was verified by PCR, Southern, northern and western blots. Mice were immunized both intraperitoneally (i.p.) and orally with tomato protein extracts containing the N protein induced the production of antibodies. The antibody titer of mice immunized i.p., was at least four times higher than that of mice immunized orally. These results were reflected in the challenge experiments where i.p.-immunized mice were partially protected against a peripheral virus challenge whereas orally immunized mice were not. This protection was comparable to that obtained in previous experiments employing different expression systems. Work is in progress to express both G and N proteins in transgenic plants and evaluate protection in mice.     

Chloroplast Genetic Engineering: Recent Advances and Future Perspectives

Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.     

HIV-1 p24-immunoglobulin fusion molecule: a new strategy for plant-based protein production

We describe the engineering of a human immunodeficiency virus-1 (HIV-1) p24-immunoglobulin A (IgA) antigen-antibody fusion molecule for therapeutic purposes and its enhancing effect on fused antigen expression in tobacco plants. Although many recombinant proteins have been expressed in transgenic plants as vaccine candidates, low levels of expression are a recurring problem. In this paper, using the HIV p24 core antigen as a model vaccine target, we describe a strategy for increasing the yield of a recombinant protein in plants. HIV p24 antigen was expressed as a genetic fusion with the alpha2 and alpha3 constant region sequences from human Ig alpha-chain and targeted to the endomembrane system. The expression of this fusion protein was detected at levels approximately 13-fold higher than HIV p24 expressed alone, and a difference in the behaviour of the two recombinant proteins during trafficking in the plant secretory pathway has been identified. Expressing the antigen within the context of alpha-chain Ig sequences resulted in the formation of homodimers and the antigen was correctly recognized by specific antibodies. Furthermore, the HIV p24 elicited T-cell and antibody responses in immunized mice. The use of Ig fusion partners is proposed as a generic platform technology for up-regulating the expression of antigens in plants, and may represent the first step in a strategy to design new vaccines with enhanced immunological properties.     

Plants as bioreactors for the production of vaccine antigens

Plants have been identified as promising expression systems for commercial production of vaccine antigens. In phase I clinical trials several plant-derived vaccine antigens have been found to be safe and induce sufficiently high immune response. Thus, transgenic plants, including edible plant parts are suggested as excellent alternatives for the production of vaccines and economic scale-up through cultivation. Improved understanding of plant molecular biology and consequent refinement in the genetic engineering techniques have led to designing approaches for high level expression of vaccine antigens in plants. During the last decade, several efficient plant-based expression systems have been examined and more than 100 recombinant proteins including plant-derived vaccine antigens have been expressed in different plant tissues. Estimates suggest that it may become possible to obtain antigen sufficient for vaccinating millions of individuals from one acre crop by expressing the antigen in seeds of an edible legume, like peanut or soybean. In the near future, a plethora of protein products, developed through ‘naturalized bioreactors’ may reach market. Efforts for further improvements in these technologies need to be directed mainly towards validation and applicability of plant-based standardized mucosal and edible vaccines, regulatory pharmacology, formulations and the development of commercially viable GLP protocols. This article reviews the current status of developments in the area of use of plants for the development of vaccine antigens.     

Production of recombinant allergens in plants

A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.     

Foreign protein production using plant cell and organ cultures: Advantages and limitations

Plants and plant tissue cultures are used as host systems for expression of foreign proteins including antibodies, vaccines and other therapeutic agents. Recombinant or stably transformed plants and plant cell cultures have been applied for foreign protein production for about 20 years. Because the product concentration achieved exerts a major influence on process economics, considerable efforts have been made by commercial and academic research groups to improve foreign protein expression levels. However, post-synthesis product losses due to protease activity within plant tissues and/or extracellular protein adsorption in plant cell cultures can negate the benefits of molecular or genetic enhancement of protein expression. Transient expression of foreign proteins using plant viral vectors is also a practical approach for producing foreign proteins in plants. Adaptation of this technology is required to allow infection and propagation of engineered viruses in plant tissue cultures for transient protein expression in vitro.     

Molecular farming of recombinant antibodies in plants.

'Molecular farming' is the production of recombinant proteins in plants. It is intended to harness the power of agriculture to cultivate and harvest transgenic plants producing recombinant therapeutics. Molecular farming has the potential to provide virtually unlimited quantities of recombinant antibodies for use as diagnostic and therapeutic tools in both health care and the life sciences. Importantly, recombinant antibody expression can be used to modify the inherent properties of plants, for example by using expressed antipathogen antibodies to increase disease resistance. Plant transformation is technically straightforward for model plant species and some cereals, and the functional expression of recombinant proteins can be rapidly analyzed using transient expression systems in intact or virally infected plants. Protein production can then be increased using plant suspension cell production in fermenters, or by the propagation of stably transformed plant lines in the field. Transgenic plants can be exploited to produce organs rich in a recombinant protein for its long-term storage. This demonstrates the promise of using transgenic plants as bioreactors for the 'molecular farming' of recombinant therapeutics, blood substitutes and diagnostics, such as recombinant antibodies.     

Development of chloroplast transformation vectors, and a new target region in the tobacco plastid genome

Plastid transformation vectors are used for high-level expression of industrially important recombinant proteins in plants. In the present study, new vectors for plastid transformation were developed. One of these vectors targets transgenes at a new site in the chloroplast genome. Intergenic regions of trnfM-trnG, ndhB-trnL and rrn16-trnV were selected as sites for transgene insertion. Tobacco chloroplast was successfully transformed with designed vectors, and the transplastomic plants accumulated recombinant protein as high as 5–6% of total soluble protein which remained localized in the chloroplasts. Although the vectors were designed using the plastid genome of Nicotiana tabacum, flanking regions used in two vectors show a high level of homology with chloroplast genomes of other plant species, thus it might be possible to use them for the transformation of a wider range of plant species.     

Modifying fatty acid profiles through a new cytokinin‐based plastid transformation system

The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin‐based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3‐ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over‐expression of isopentenyl transferase. Over‐expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic‐based selection systems, and may be used in combination with a negative selectable marker as part of a two‐step selection system to obtain homoplasmic plant lines.     

High‐level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes

Transgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance. In this article, we explore the potential of transplastomic plants to produce human immunodeficiency virus (HIV) antigens as potential components of an acquired immunodeficiency syndrome (AIDS) vaccine. It is shown that the HIV antigens p24 (the major target of T‐cell‐mediated immune responses in HIV‐positive individuals) and Nef can be expressed to high levels in plastids of tobacco, a non‐food crop, and tomato, a food crop with an edible fruit. Optimized p24‐Nef fusion gene cassettes trigger antigen protein accumulation to up to approximately 40% of the plant's total protein, demonstrating the great potential of transgenic plastids to produce AIDS vaccine components at low cost and high yield.     

Codon optimization of <Emphasis Type="Italic">cry1Ab</Emphasis> gene for hyper expression in plant organelles

With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome, following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well.     

The expression of classical swine fever virus structural protein E2 gene in tobacco chloroplasts for applying chloroplasts as bioreactors

It has been reported that genes encoding antigens of bacterial and viral pathogens can be expressed in plants and are shown to induce protection antibodies. The structural protein E2 of classical swine fever virus (CSFV), which has been shown to carry critical epitopes, has been expressed in different systems. Here, we report the expression of CFSV E2 gene in tobacco chloroplasts. Mice immunized with leaf extracts elicited specific antibodies. This indicated that the expressed E2 proteins had a certain degree of immunogenicity. To our knowledge, this is the first report showing induction of protective antibody in response to classical swine fever virus (CSFV) by immunization with antigen protein E2 expressed in tobacco chloroplasts, which will open a new way to protection from CSFV by plant chloroplasts as bioreactors.     

Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit

Transgenic chloroplasts offer unique advantages in plant biotechnology, including high-level foreign protein expression, absence of epigenetic effects, and gene containment due to the lack of transgene transmission through pollen. However, broad application of plastid genome engineering in biotechnology has been largely hampered by both the lack of chloroplast transformation systems for major crop plants and the usually low plastid gene expression levels in nongreen tissues such as fruits, tubers, and other storage organs. Here we describe the development of a plastid transformation system for tomato, Lycopersicon esculentum. This is the first report on the generation of fertile transplastomic plants in a food crop with an edible fruit. We show that chromoplasts in the tomato fruit express the transgene to approximately 50% of the expression levels in leaf chloroplasts. Given the generally very high foreign protein accumulation rates that can be achieved in transgenic chloroplasts (>40% of the total soluble protein), this system paves the way to efficient production of edible vaccines, pharmaceuticals, and antibodies in tomato.     

OBPC Symposium: Maize 2004 &; beyond—Recent advances in chloroplast genetic engineering

Summary The chloroplast genetic engineering approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. Thus far, more than 40 transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer several agronomic traits and produce vaccine antigens, industrially valuable enzymes, biomaterials, and amino acids. Functionality of chloroplastderived of vaccine antigens has been facilitated by hyperexpression in transgenic chloroplasts (leaves) or non-green plastids (carrols) and the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Despite such significant progress in chloroplast transformation, this technology has not been extended to major crops. This obstacle emphasizes the need for plastid genome sequencing to increase the efficiency of transformation and conduct basic research in plastid biogenesis and function. However, highly efficient soybean, carrot, and cotton plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors. Recent advancements facilitate our understanding of plastid biochemistry and molecular biology. This review focuses on exciting recent developments in this field and offers directions for further research and development.