Pascal M. W. Drake

Genetically modified plants and human health

Summary Genetically modified (or GM) plants have attracted a large amount of media attention in recent years and continue to do so. Despite this, the general public remains largely unaware of what a GM plant actually is or what advantages and disadvantages the technology has to offer, particularly with regard to the range of applications for which they can be used. From the first generation of GM crops, two main areas of concern have emerged, namely risk to the environment and risk to human health. As GM plants are gradually being introduced into the European Union there is likely to be increasing public concern regarding potential health issues. Although it is now commonplace for the press to adopt ‘health campaigns’, the information they publish is often unreliable and unrepresentative of the available scientific evidence. We consider it important that the medical profession should be aware of the state of the art, and, as they are often the first port of call for a concerned patient, be in a position to provide an informed opinion. This review will examine how GM plants may impact on human health both directly – through applications targeted at nutrition and enhancement of recombinant medicine production – but also indirectly, through potential effects on the environment. Finally, it will examine the most important opposition currently facing the worldwide adoption of this technology: public opinion.

Highly Immunogenic and Protective Recombinant Vaccine Candidate Expressed in Transgenic Plants

ABSTRACT Vaccine development has been hampered by difficulties in developing new and safe adjuvants, so alternative technologies that offer new avenues forward are urgently needed. The goal of this study was to express a monoclonal recombinant immune complex in a transgenic plant. A recombinant protein consisting of a tetanus toxin C fragment-specific monoclonal antibody fused with the tetanus toxin C fragment was designed and expressed. Immune complex formation occurred between individual fusion proteins to form immune complex-like aggregates that bound C1q and FcγRIIa receptor and could be targeted to antigen-presenting cells. Unlike antigen alone, the recombinant immune fusion complexes were highly immunogenic in mice and did not require coadministration of an adjuvant (when injected subcutaneously). Indeed, these complexes elicited antibody titers that were more than 10,000 times higher than those observed in animals immunized with the antigen alone. Furthermore, animals immunized with only 1 μg of recombinant immune complex without adjuvant were fully protected against lethal challenge. This the first report on the use of a genetic fusion between antigen and antibody to ensure an optimal expression ratio between the two moieties and to obtain fully functional recombinant immune complexes as a new vaccine model.

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 μg/ml hydroponic media after 24 days. Therefore, we suggest that transgenic plants have the potential to provide strategies for large‐scale protein microbicide production.

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 plantderived 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 Guys 13 and 4E10 and one expressing a small microbicide polypeptide cyanovirin‐N. Rhizosecretion rates increased significantly by the addition of the plant growth regulator α‐naphthalene acetic acid. The maximum rhizosecretion rates achieved were 58 Hg/g root dry weight/24 h for Guys 13, 10.43 μg/g root dry weight/24 h for 4E10, and 766 μg/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 indolebutyric acid, 6–benzylaminopurine, and kinetin were also demonstrated to increase rhizosecretion of Guys 13. The effect of the growth regulators differed, as α‐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 Guys 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 Guys 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 Guys 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.—Drake, P. M. W., Barbi, T., Sexton, A., McGowan, E., Stadlmann, J., Navarre, C., Paul, M. J., Ma, J. K.‐C. Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco. FASEB J. 23, 3581–3589 (2009). www.fasebj.org

Antibody degradation in tobacco plants: a predominantly apoplastic process

BackgroundInterest 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.ResultsIn this study, we investigated the dynamics of the assembly and breakdown of a human IgG1κ 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.ConclusionsThe 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.

Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV

Recombinant Secretory IgA (SIgA) complexes have the potential to improve antibody-based passive immunotherapeutic approaches to combat many mucosal pathogens. In this report, we describe the expression, purification and characterization of a human SIgA format of the broadly neutralizing anti-HIV monoclonal antibody (mAb) 2G12, using both transgenic tobacco plants and transient expression in Nicotiana benthamiana as expression hosts (P2G12 SIgA). The resulting heterodecameric complexes accumulated in intracellular compartments in leaf tissue, including the vacuole. SIgA complexes could not be detected in the apoplast. Maximum yields of antibody were 15.2 μg/g leaf fresh mass (LFM) in transgenic tobacco and 25 μg/g LFM after transient expression, and assembly of SIgA complexes was superior in transgenic tobacco. Protein L purified antibody specifically bound HIV gp140 and neutralised tier 2 and tier 3 HIV isolates. Glycoanalysis revealed predominantly high mannose structures present on most N-glycosylation sites, with limited evidence for complex glycosylation or processing to paucimannosidic forms. O-glycan structures were not identified. Functionally, P2G12 SIgA, but not IgG, effectively aggregated HIV virions. Binding of P2G12 SIgA was observed to CD209 / DC-SIGN, but not to CD89 / FcalphaR on a monocyte cell line. Furthermore, P2G12 SIgA demonstrated enhanced stability in mucosal secretions in comparison to P2G12 IgG mAb.

Immune-Complex Mimics as a Molecular Platform for Adjuvant-Free Vaccine Delivery

Protein-based vaccine development faces the difficult challenge of finding robust yet non-toxic adjuvants suitable for humans. Here, using a molecular engineering approach, we have developed a molecular platform for generating self-adjuvanting immunogens that do not depend on exogenous adjuvants for induction of immune responses. These are based on the concept of Immune Complex Mimics (ICM), structures that are formed between an oligomeric antigen and a monoclonal antibody (mAb) to that antigen. In this way, the roles of antigens and antibodies within the structure of immune complexes are reversed, so that a single monoclonal antibody, rather than polyclonal sera or expensive mAb cocktails can be used. We tested this approach in the context of Mycobacterium tuberculosis (MTB) infection by linking the highly immunogenic and potentially protective Ag85B with the oligomeric Acr (alpha crystallin, HspX) antigen. When combined with an anti-Acr monoclonal antibody, the fusion protein formed ICM which bound to C1q component of the complement system and were readily taken up by antigen-presenting cells in vitro. ICM induced a strong Th1/Th2 mixed type antibody response, which was comparable to cholera toxin adjuvanted antigen, but only moderate levels of T cell proliferation and IFN-γ secretion. Unfortunately, the systemic administration of ICM did not confer statistically significant protection against intranasal MTB challenge, although a small BCG-boosting effect was observed. We conclude that ICM are capable of inducing strong humoral responses to incorporated antigens and may be a suitable vaccination approach for pathogens other than MTB, where antibody-based immunity may play a more protective role.

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 IgG1κ, Guys 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 µg/g leaf fresh weight (0.6% total soluble protein), and the rate of antibody rhizosecretion reached a maximum of 5 µg/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. FASEB J. 24, 882–890 (2010). www.fasebj.org

Rhizosecretion improves the production of Cyanovirin-N in Nicotiana tabacum through simplified downstream processing.

Rhizosecretion has many advantages for the production of recombinant pharmaceuticals, notably facile downstream processing from hydroponic medium. The aim of this study was to increase yields of the HIV microbicide candidate, Cyanovirin-N (CV-N), obtained using this production platform and to develop a simplified methodology for its downstream processing from hydroponic medium. Placing hydroponic cultures on an orbital shaker more than doubled the concentration of CV-N in the hydroponic medium compared to plants which remained stationary, reaching a maximum of approximately 20μg/ml in one week, which is more than 3 times higher than previously reported yields. The protein composition of the hydroponic medium, the rhizosecretome, was characterised in plants cultured with or without the plant growth regulator alpha-napthaleneacetic acid by LC-ESI-MS/MS, and CV-N was the most abundant protein. The issue of large volumes in the rhizosecretion system was addressed by using ion exchange chromatography to concentrate CV-N and partially remove impurities. The semi-purified CV-N was demonstrated to bind to HIV gp120 in an ELISA and to neutralise HIVBa-L with an IC50 of 6nM in a cell-based assay. Rhizosecretion is therefore a practicable and inexpensive method for the production of functional CV-N.

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

HighlightsMurine version of IL‐4&Dgr;2 appears to be fully functional.IL‐4&Dgr;2 does not compete with IL‐4 for IL‐4R&agr; binding.IL‐4&Dgr;2 does not interfere with STAT‐6 signalling.Alternative, yet unknown mechanism of IL‐4&Dgr;2 modulating IL‐4 activity must exist. Abstract IL‐4 is a pleiotropic cytokine that is highly Th2 polarizing. The ratio of IL‐4 and its splice variant IL‐4&Dgr;2 observed in human health and disease suggests a role for both isoforms. In the present study, the biological function of murine IL‐4&Dgr;2 and the potential mechanism of action were studied. We report for the first time the generation of a functional, recombinant murine IL‐4&Dgr;2 form which is suggestive of its possible biological role in this species. Recombinant murine IL‐4&Dgr;2 inhibited IL‐4 mediated cellular processes in macrophages and lymphocytes. Specifically, (i) it reversed IL‐4 mediated inhibition of IFN‐&ggr; 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‐4&Dgr;2 did not compete with IL‐4 for IL‐4R&agr; 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.