Multiple gene expression in plants using MIDAS-P, a versatile type II restriction-based modular expression vector.

MIDAS-P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly arranged transcription units (TUs). We have used the MIDAS-P system to investigate the expression of up to five genes encoding three anti-HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti-HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single-chain camelid nanobody (J3-VHH). Constructs containing different combinations of 3, 4, or 5 TUs encoding different components of the anti-HIV cocktail were assembled. Messenger RNA (mRNA) levels of the genes of interest decreased beyond two TUs. Coexpression of the RNA silencing suppressor P19 dramatically increased the overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non-detectable levels in constructs with four or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co-expression of P19. In short, the MIDAS-P system is suitable for the simultaneous expression of multiple proteins in one construct.

Rational design and expression of a recombinant plant rhabdovirus glycoprotein for production of immunoreactive murine anti-sera.

Lettuce necrotic yellows virus (LNYV) is a plant rhabdovirus which has a type-1 transmembrane glycoprotein. Here, we describe the generation of murine anti-sera to the glycoprotein. Rational design, expression, and purification of recombinant glycoprotein, termed rLGe, was undertaken using SignalP4.1 and camSol servers to predict signal peptide cleavage and protein solubility. In order to successfully obtain expression in mammalian cells, LNYV glycoprotein native signal peptide was substituted with that of Rabies virus glycoprotein. In addition, rather than expression of the entire molecule, rLGe consisted of the LNYV glycoprotein ectodomain fused to two affinity tags to minimize the risk of protein aggregation, while facilitating detection and purification. rLGe was transiently expressed in mammalian cell culture, purified using affinity column chromatography, and used to immunize mice. Harvested anti-sera were immunoreactive and specific to the naturally occurring glycoprotein as confirmed by western blotting of plant leaf tissue infected with LNYV.

Murine IL-4Δ2 splice variant down-regulates IL-4 activities independently of IL-4Rα binding and STAT-6 phosphorylation.

IL-4 is a pleiotropic cytokine that is highly Th2 polarizing. The ratio of IL-4 and its splice variant IL-4Δ2 observed in human health and disease suggests a role for both isoforms. In the present study, the biological function of murine IL-4Δ2 and the potential mechanism of action were studied. We report for the first time the generation of a functional, recombinant murine IL-4Δ2 form which is suggestive of its possible biological role in this species. Recombinant murine IL-4Δ2 inhibited IL-4 mediated cellular processes in macrophages and lymphocytes. Specifically, (i) it reversed IL-4 mediated inhibition of IFN-γ induced nitric oxide release by macrophages, (ii) inhibited IL-4 mediated induction of T cell proliferation, and (iii) prevented IL-4 stimulation of IgE synthesis by B cells. However, IL-4Δ2 did not compete with IL-4 for IL-4Rα binding and did not interfere with the downstream STAT-6 phosphorylation in T cells, suggesting an alternative mechanism for its antagonism of specific IL4-driven effects. These findings suggest that the mouse is a suitable experimental model for studies of the biology of IL-4 and its alternative splice variant.

Recombinant biologic products versus nutraceuticals from plants - a regulatory choice?

Biotechnology has transformed the potential for plants to be a manufacturing source of pharmaceutical compounds. Now, with transgenic and transient expression techniques, virtually any biologic, including vaccines and therapeutics, could be manufactured in plants. However, uncertainty over the regulatory path for such new pharmaceuticals has been a deterrent. Consideration has been given to using alternative regulatory paths, including those for nutraceuticals or cosmetic agents. This review will consider these possibilities, and discuss the difficulties in establishing regulatory guidelines for new pharmaceutical manufacturing technologies.

High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 µg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies.

Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

Transformation of Althaea officinalis L. by Agrobacterium rhizogenes for the production of transgenic roots expressing the anti-HIV microbicide cyanovirin-N.

The marshmallow plant (Althaea officinalis L.) has been used for centuries in medicine and other applications. Valuable secondary metabolites have previously been identified in Agrobacterium rhizogenes-generated transgenic 'hairy' roots in this species. In the present study, transgenic roots were produced in A. officinalis using A. rhizogenes. In addition to wild-type lines, roots expressing the anti-human immunodeficiency virus microbicide candidate, cyanovirin-N (CV-N), were generated. Wild-type and CV-N root lines were transferred to liquid culture and increased in mass by 49 and 19 % respectively over a 7 day culture period. In the latter, the concentration of CV-N present in the root tissue was 2.4 µg/g fresh weight, with an average secretion rate into the growth medium of 0.02 µg/ml/24 h. A. officinalis transgenic roots may therefore in the future be used not only as a source of therapeutic secondary metabolites, but also as an expression system for the production of recombinant pharmaceuticals.

Production of Recombinant Proteins in Transgenic Tobacco Plants.

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.

Antibody degradation in tobacco plants: a predominantly apoplastic process.

BACKGROUND: Interest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem. RESULTS: In this study, we investigated the dynamics of the assembly and breakdown of a human IgG(1)κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space. CONCLUSIONS: The results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.

Expression and plasma membrane localization of the mammalian B-cell receptor complex in transgenic Nicotiana tabacum.

The B-cell antigen receptor (BCR), displayed on the plasma membrane of mature B cells of the mammalian immune system, is a multimeric complex consisting of a membrane-bound immunoglobulin (mIg) noncovalently associated with the Igα/Igß heterodimer. In this study, we engineered transgenic tobacco plants expressing all four chains of the BCR. ELISA, Western blotting and confocal microscopy demonstrated that the BCR was correctly assembled in plants, predominantly in the plasma membrane, and that the noncovalent link was detergent sensitive. This is the first example of a noncovalently assembled plasma membrane-retained heterologous receptor in plants. In B cells of the mammalian immune system, following antigen binding to mIg, BCR is internalized and tyrosine residues on Igα and Igß are phosphorylated activating a signaling cascade through interaction with protein kinases that ultimately leads to the initiation of gene expression. Expression of the BCR may therefore be an important tool for the study of plant endocytosis and the identification of previously unknown plant tyrosine kinases. The specificity and diversity of the antibody repertoire, coupled to the signal transduction capability of the Igα/Igß heterodimer, also indicates that plants expressing BCR may in future be developed as environmental biosensors.

Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

We describe the engineering, regeneration, and characterization of transgenic tobacco plants expressing a recombinant antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by species of cyanobacteria. The antibody was created by a genetic fusion of the antigen-binding regions of the microcystin-specific single-chain antibody, 3A8, with constant regions from the murine IgG1kappa, Guy's 13. IgG transgenes were controlled by a leader peptide that targets the transgene products to the secretory pathway and also allows for rhizosecretion. The antibody, extracted from the leaves or rhizosecreted into hydroponic medium by transgenic plants, was shown to have functional binding to MC-LR. Antibody yields in transgenic plant leaves reached a maximum of 64 microg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 microg/g root dry weight/24 h. Rhizosecreted antibody bound to MC-LR in hydroponic medium, and transgenic plants grew more efficiently on medium containing MC-LR compared to wild-type controls. This proof of concept paves the way for applications to produce diagnostic antibodies to microcystin-LR, remove it from the environment by phytoremediation, or enhance yields in crops exposed to MC-LR.-Drake, P. M. W., Barbi, T., Drever, M. R., van Dolleweerd, C. J., Porter, A. J. R., Ma, J. K.-C. Generation of transgenic plants expressing antibodies to the environmental pollutant microcystin-LR.

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR.

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy's 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 µg g(-1) leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco.

Rhizosecretion is an attractive technology for the production of recombinant proteins from transgenic plants. However, to date, yields of plant-derived recombinant pharmaceuticals by this method have been too low for commercial viability. Studies conducted focused on three transgenic plant lines grown in hydroponic culture medium, two expressing monoclonal antibodies Guy's 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin-N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator alpha-naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 microg/g root dry weight/24 h for Guy's 13, 10.43 microg/g root dry weight/24 h for 4E10, and 766 microg/g root dry weight/24 h for cyanovirin-N, the highest figures so far reported for a full-length antibody and a recombinant protein, respectively. The plant growth regulators indole-butyric acid, 6-benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guy's 13. The effect of the growth regulators differed, as alpha-naphthalene acetic acid and indole-butyric acid increased the root dry weight of hydroponic plants, whereas the cytokinins benzylaminopurine and kinetin increased rhizosecretion without affecting root mass. A comparative glycosylation analysis between MAb Guy's 13 purified from either hydroponic culture medium or from leaf extracts demonstrated a similar pattern of glycosylation comprising high mannose to complex glycoforms. Analysis of the hydroponic culture medium at harvest revealed significantly lower and less complex levels of proteolytic enzymes, in comparison with leaf extracts, which translated to a higher proportion of intact Guy's 13 IgG in relation to other IgG products. Hydroponic medium could be added directly to a chromatography column for affinity purification, allowing simple and rapid production of high purity Guy's 13 antibody. In addition to the attractiveness of controlled cultivation within a contained environment for pharmaceutical-producing plants, this study demonstrates advantages with respect to the quality and downstream purification of recombinant proteins.

Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals.

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.

Development of a minigenome cassette for Lettuce necrotic yellows virus: A first step in rescuing a plant cytorhabdovirus.

Rhabdoviruses are enveloped negative-sense RNA viruses that have numerous biotechnological applications. However, recovering plant rhabdoviruses from cDNA remains difficult due to technical difficulties such as the need for concurrent in planta expression of the viral genome together with the viral nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L) and viral genome instability in E. coli. Here, we developed a negative-sense minigenome cassette for Lettuce necrotic yellows virus (LNYV). We introduced introns into the unstable viral ORF and employed Agrobacterium tumefaciens to co-infiltrate Nicotiana with the genes for the N, P, and L proteins together with the minigenome cassette. The minigenome cassette included the Discosoma sp. red fluorescent protein gene (DsRed) cloned in the negative-sense between the viral trailer and leader sequences which were placed between hammerhead and hepatitis delta ribozymes. In planta DsRed expression was demonstrated by western blotting while the appropriate splicing of introduced introns was confirmed by sequencing of RT-PCR product.

Mucosal Therapy of Multi-Drug Resistant Tuberculosis With IgA and Interferon-γ.

New evidence has been emerging that antibodies can be protective in various experimental models of tuberculosis. Here, we report on protection against multidrug-resistant Mycobacterium tuberculosis (MDR-TB) infection using a combination of the human monoclonal IgA 2E9 antibody against the alpha-crystallin (Acr, HspX) antigen and mouse interferon-gamma in mice transgenic for the human IgA receptor, CD89. The effect of the combined mucosal IgA and IFN-γ; treatment was strongest (50-fold reduction) when therapy was applied at the time of infection, but a statistically significant reduction of lung bacterial load was observed even when the therapy was initiated once the infection had already been established. The protection involving enhanced phagocytosis and then neutrophil mediated killing of infected cells was IgA isotype mediated, because treatment with an IgG version of 2E9 antibody was not effective in human IgG receptor CD64 transgenic mice. The Acr antigen specificity of IgA antibodies for protection in humans has been indicated by their elevated serum levels in latent tuberculosis unlike the lack of IgA antibodies against the virulence-associated MPT64 antigen. Our results represent the first evidence for potential translation of mucosal immunotherapy for the management of MDR-TB.

Shotguns vs Lasers: Identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products.

Plant molecular farming (PMF) is a convenient and cost-effective way to produce high-value recombinant proteins that can be used in the production of a range of health products, from pharmaceutical therapeutics to cosmetic products. New plant breeding techniques (NPBTs) provide a means to enhance PMF systems more quickly and with greater precision than ever before. However, the feasibility, regulatory standing and social acceptability of both PMF and NPBTs are in question. This paper explores the perceptions of key stakeholders on two European Union (EU) Horizon 2020 programmes-Pharma-Factory and Newcotiana-towards the barriers and facilitators of PMF and NPBTs in Europe. One-on-one qualitative interviews were undertaken with N = 20 individuals involved in one or both of the two projects at 16 institutions in seven countries (Belgium, France, Germany, Italy, Israel, Spain and the UK). The findings indicate that the current EU regulatory environment and the perception of the public towards biotechnology are seen as the main barriers to scaling-up PMF and NPBTs. Competition from existing systems and the lack of plant-specific regulations likewise present challenges for PMF developing beyond its current niche. However, respondents felt that the communication of the benefits and purpose of NPBT PMF could provide a platform for improving the social acceptance of genetic modification. The importance of the media in this process was highlighted. This article also uses the multi-level perspective to explore the ways in which NPBTs are being legitimated by interested parties and the systemic factors that have shaped and are continuing to shape the development of PMF in Europe.

Transgenic plant production of Cyanovirin-N, an HIV microbicide.

Cyanovirin-N (CV-N) is a microbicide candidate that inactivates a wide range of HIV strains by binding to gp120. Production of CV-N, or any protein microbicide, needs to be at extremely high levels and low cost to have an impact on global health. Thus, it is unlikely that fermentor-based systems will be suitable, including recombinant E. coli, where CV-N aggregates and dimers have consistently been found. Transgenic plants may provide a suitable expression system for protein microbicides, as production can be easily and economically scaled up. Here, Nicotiana tabacum was transformed with a gene encoding CV-N to explore proof of concept for the production of CV-N in transgenic plants. Plant-derived rCV-N was recoverable at levels of 130 ng/mg of fresh leaf tissue, or at least 0.85% of total soluble plant protein. Western blot analysis demonstrated that virtually all of the rCV-N was expressed in the desired monomeric form. Functionality was demonstrated by specific binding to gp120 and protection of T-cells from in vitro HIV infection. Hydroponic culturing of transgenic plants demonstrated CV-N rhizosecretion at levels of 0.64 mug/ml hydroponic media after 24 days. Therefore, we suggest that transgenic plants have the potential to provide strategies for large-scale protein microbicide production.

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.